U.S. patent application number 10/409611 was filed with the patent office on 2004-12-23 for method of enhancing of binding activity of antibody composition to fcgamma receptor iiia.
This patent application is currently assigned to KYOWA HAKKO KOGYO CO., LTD.. Invention is credited to Nakamura, Kazuyasu, Shitara, Kenya.
Application Number | 20040259150 10/409611 |
Document ID | / |
Family ID | 28786448 |
Filed Date | 2004-12-23 |
United States Patent
Application |
20040259150 |
Kind Code |
A1 |
Nakamura, Kazuyasu ; et
al. |
December 23, 2004 |
Method of enhancing of binding activity of antibody composition to
Fcgamma receptor IIIa
Abstract
A method for enhancing a binding activity of an antibody
composition to Fc.gamma. receptor IIIa, which comprises modifying a
complex N-glycoside-linked sugar chain which is bound to the Fc
region of an antibody molecule; a method for enhancing an
antibody-dependent cell-mediated cytotoxic activity of an antibody
composition; a process for producing an antibody composition having
an enhanced binding activity to Fc.gamma. receptor IIIa; a method
for detecting the ratio of a sugar chain in which fucose is not
bound to N-acetylglucosamine in the reducing end in the sugar chain
among total complex N-glycoside-linked sugar chains bound to the Fc
region in an antibody composition; an Fc fusion protein composition
produced by using a cell resistant to a lectin which recognizes a
sugar chain in which 1-position of fucose is bound to 6-position of
N-acetylglucosamine in the reducing end through .alpha.-bond in a
complex N-glycoside-linked sugar chain; and a process for producing
the same.
Inventors: |
Nakamura, Kazuyasu; (Tokyo,
JP) ; Shitara, Kenya; (Tokyo, JP) |
Correspondence
Address: |
NIXON & VANDERHYE P.C.
8th Floor
1100 North Globo Rd.
Arlington
VA
22201-4714
US
|
Assignee: |
KYOWA HAKKO KOGYO CO., LTD.
Tokyo
JP
|
Family ID: |
28786448 |
Appl. No.: |
10/409611 |
Filed: |
April 9, 2003 |
Current U.S.
Class: |
435/7.1 ;
435/68.1 |
Current CPC
Class: |
A61P 35/00 20180101;
A61P 37/04 20180101; C07K 2317/41 20130101; C12P 21/02 20130101;
A61P 29/00 20180101; A61P 37/08 20180101; C07K 2317/732 20130101;
A61K 39/395 20130101; C07K 16/283 20130101; A61P 31/14 20180101;
A61P 31/04 20180101; A61P 37/02 20180101; A61P 43/00 20180101; C07K
2319/30 20130101; A61P 9/00 20180101; A61P 31/12 20180101; C07K
16/22 20130101; C07K 16/3084 20130101; C07K 16/2866 20130101; C07K
2317/24 20130101 |
Class at
Publication: |
435/007.1 ;
435/068.1 |
International
Class: |
G01N 033/53; C12P
021/06 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 9, 2002 |
JP |
P.2002-106950 |
Claims
1. A method for enhancing a binding activity of an antibody
composition to Fc.gamma. receptor IIIa, which comprises modifying a
complex N-glycoside-linked sugar chain which is bound to the Fc
region of an antibody molecule.
2. The method according to claim 1, wherein the modification of a
complex N-glycoside-linked sugar chain which is bound to the Fc
region of an antibody molecule is to bind a sugar chain in which
1-position of fucose is not bound to 6-position of
N-acetylglucosamine in the reducing end through .alpha.-bond in the
complex N-glycoside-linked sugar chain to the Fc region in the
antibody molecule.
3. The method according to claim 1, wherein the sugar chain is
synthesized by a cell in which the activity of a protein relating
to modification of a sugar chain in which fucose is bound to
N-acetylglucosamine in the reducing end in the complex
N-glycoside-linked sugar chain is decreased or deleted.
4. The method according to claim 3, wherein the protein relating to
(a) a protein relating to synthesis of an intracellular sugar
nucleotide, GDP-fucose; (b) a protein relating to modification of a
sugar chain in which 1-position of fucose is bound to 6-position of
N-acetylglucosamine in the reducing end through .alpha.-bond in a
complex N-glycoside-linked sugar chain; (c) a protein relating to
transport of an intracellular sugar nucleotide, GDP-fucose to the
Golgi body.
5. The method according to claim 3, wherein the cell is resistant
to a lectin which recognizes a sugar chain structure in which
1-position of fucose is bound to 6-position of N-acetylglucosamine
in the reducing end through .alpha.-bond in a complex
N-glycoside-linked sugar chain.
6. The method according to claim 3, wherein the cell is resistant
to at least one lectin selected from the group consisting of the
following (a) to (d): (a) a Lens culinaris lectin;
7. The method according to claim 3, wherein the cell is selected
from the group consisting of a yeast, an animal cell, an insect
cell and a plant cell.
8. The method according to claim 3, wherein the cell is selected
from the group consisting of the following (a) to (i): (a) a CHO
cell derived from a Chinese hamster ovary tissue; (b) a rat myeloma
cell line YB2/3HL.P2.G11.16Ag.20 cell; (c) a BHK cell derived from
a Syrian hamster kidney tissue; (d) a mouse myeloma cell line NS0
cell; (e) a mouse myeloma cell line SP2/0-Ag14 cell; (f) a
hybridoma cell; (g) a human leukemic cell line Namalwa cell; (h) an
embryonic stem cell; (i) a fertilized egg cell.
9. The method according to claim 1, (c) an antibody fragment
comprising the Fc region of (a) or (b); (d) a fusion protein
comprising the Fc region of (a) or (b).
10. The method according to claim 1, wherein the antibody molecule
belongs to an IgG class.
11. The method according to claim 1, wherein, in the complex
N-glycoside-linked sugar chain which is bound to the Fc region of
an antibody molecule, the ratio of a sugar chain in which
1-position of fucose is not bound to 6-position of
N-acetylglucosamine in the reducing end through .alpha.-bond is 20%
or more of total complex N-glycoside-linked sugar chains.
12. A method for enhancing an antibody-dependent cell-mediated
cytotoxic activity of an antibody composition, which comprises
using the method according to claim 1.
13. A process for producing an antibody composition having an
14. The process according to claim 13, wherein the modification of
a complex N-glycoside-linked sugar chain which is bound to the Fc
region of an antibody molecule is to bin a sugar chain in which
1-position of fucose is not bound to 6-position of
N-acetylglucosamine in the reducing end through .alpha.-bond in the
complex N-glycoside-linked sugar chain to the Fc region in the
antibody molecule.
15. The process according to claim 13, wherein the sugar chain is
synthesized by a cell in which the activity of a protein relating
to modification of a sugar chain in which fucose is bound to
N-acetylglucosamine in the reducing end in the complex
N-glycoside-linked sugar chain is decreased or deleted.
16. The process according to claim 15, wherein the protein relating
to modification of a sugar chain in which fucose is bound to
N-acetylglucosamine in the reducing end in the complex
N-glycoside-linked sugar chain is selected from the group
consisting of the following (a), (b) and (c): (a) a protein
relating to synthesis of an intracellular sugar nucleotide,
GDP-fucose; (c) a protein relating to transport of an intracellular
sugar nucleotide, GDP-fucose to the Golgi body.
17. The process according to claim 15, wherein the cell is
resistant to a lectin which recognizes a sugar chain structure in
which 1-position of fucose is bound to 6-position of
N-acetylglucosamine in the reducing end through .alpha.-bond in a
complex N-glycoside-linked sugar chain.
18. The process according to claim 15, wherein the cell is
resistant to at least one lectin selected from the group consisting
of the following (a) to (d): (a) a Lens culinaris lectin; (b) a
Pisum sativum lectin; (c) a Vicia faba lectin; (d) an Aleuria
aurantia lectin.
19. The process according to claim 15
20. The process according to claim 15, wherein the cell is selected
from the group consisting of the following (a) to (i): (a) a CHO
cell derived from a Chinese hamster ovary tissue; (b) a rat myeloma
cell line YB2/3HL.P2.G11.16Ag.20 cell; (c) a BHK cell derived from
a Syrian hamster kidney tissue; (d) a mouse myeloma cell line NS0
cell; (e) a mouse myeloma cell line SP2/0-Ag14 cell; (f) a
hybridoma cell; (g) a human leukemic cell line Namalwa cell; (h) an
embryonic stem cell; (i) a fertilized egg cell.
21. The process according to claim 13, wherein the antibody
molecule is selected from the group consisting of the following (a)
to (d): (a) a human antibody; (b) a humanized antibody;
22. The process according to claim 13, wherein the antibody
molecule belongs to an IgG class.
23. The method according to claim 13, wherein, in the complex
N-glycoside-linked sugar chain which is bound to the Fc region of
an antibody molecule, the ratio of a sugar chain in which
1-position of fucose is not bound to 6-position of
N-acetylglucosamine in the reducing end through .alpha.-bond is 20%
or more of total complex N-glycoside-linked sugar chains.
24. A process for producing an antibody composition having an
increased antibody-dependent cell-mediated cytotoxic activity,
which comprises using the process according to claim 12.
25. An antibody composition produced by the process according to
claim 13.
26. A method for detecting the ratio of a sugar chain in which with
an Fc.gamma. receptor IIIa to measure the binding activity to the
Fc.gamma. receptor IIIa; and detecting the ratio of a sugar chain
in a standard antibody composition with a standard curve showing
the binding activity to the Fc.gamma. receptor IIIa.
27. A method for detecting the antibody-dependent cell-mediated
cytotoxic activity in an antibody composition, which comprises:
reacting an antigen with a tested antibody composition to form a
complex of the antigen and the antibody composition; contacting the
complex with an Fc.gamma. receptor IIIa to measure the binding
activity to the Fc.gamma. receptor IIIa; and detecting the ratio of
a sugar chain in a standard antibody composition with a standard
curve showing the binding activity to the Fc.gamma. receptor
IIIa.
28. A method for detecting the ratio of a sugar chain in which
fucose is not bound to N-acetylglucosamine in the reducing end in
the sugar chain among total complex N-glycoside-linked sugar chains
bound to the Fc region in an antibody composition, which comprises:
contacting a tested antibody composition with a Fc.gamma. receptor
IIIa to measure the binding activity of the antibody composition to
the Fc.gamma.
29. A method for detecting the antibody-dependent cell-mediated
cytotoxic activity in an antibody composition, which comprises:
contacting a tested antibody composition with a Fc.gamma. receptor
IIIa to measure the binding activity of the antibody composition to
the Fc.gamma. receptor IIIa; and detecting the ratio of a sugar
chain in a standard antibody composition with a standard curve
showing the binding activity to the Fc.gamma. receptor IIIa.
30. An Fc fusion protein composition produced by using a cell
resistant to a lectin which recognizes a sugar chain structure in
which 1-position of fucose is bound to 6-position of
N-acetylglucosamine in the reducing end through .alpha.-bond in a
complex N-glycoside-linked sugar chain.
31. The Fc fusion protein composition according to claim 30,
wherein the cell is selected from the group consisting of the
following (a), (b) and (c): (a) an enzyme protein relating to
synthesis of an intracellular sugar nucleotide, GDP-fucose; (b) an
enzyme protein relating to modification of a sugar chain in which
1- wherein the activity of the protein is decreased or deleted.
32. The Fc fusion protein composition according to claim 30,
wherein the cell is resistant to at least one lectin selected from
the group consisting of the following (a) to (d): (a) a Lens
culinaris lectin; (b) a Pisum sativum lectin; (c) a Vicia faba
lectin; (d) an Aleuria aurantia lectin.
33. The Fc fusion protein composition according to claim 30,
wherein the cell is a cell into which a gene encoding an Fc fusion
protein is introduced.
34. The Fc fusion protein composition according to claim 33,
wherein the Fc is derived from an IgG class of an antibody
molecule.
36. The Fc fusion protein composition according to claim 30,
wherein the cell is a mouse myeloma cell.
37. The Fc fusion protein composition according to claim 36,
wherein the mouse myeloma cell is NS0 cell or SP2/0-Ag14 cell.
38. The Fc fusion protein composition according to claim 30,
wherein the cell is selected from the group consisting of the
following (a) to (g): (a) a CHO cell derived from a Chinese hamster
ovary tissue; (b) a rat myeloma cell line YB2/3HL.P2.G11.16Ag.20
line; (c) a BHK cell derived from a Syrian hamster kidney tissue;
(d) hybridoma cell; (e) a human leukemic cell line Namalwa cell;
(f) an embryonic stem cell; (g) a fertilized egg cell.
39. An Fc fusion protein composition comprisng an Fc fusion protein
having an complex N-glycoside-linked sugar chain at the Fc region
of an antibody molecule, wherein the ratio of a sugar chain in
which fucose is not bonded to N-acetylglucosamine in the reducing
end in the sugar chain is 20% or more of total complex
N-glycoside-linked sugar chains which are bound to the Fc region in
the composition.
40. The Fc fusion protein composition according to claim 39,
wherein the sugar chain in which fucose is not bound is a sugar
chain in which 1-position of fucose is not bound to 6-position of
N-acetylglucosamine in the reducing end in the complex
N-glycoside-linked sugar chain through .alpha.-bond.
41. The Fc fusion protein composition according to claim 39,
wherein the antibody molecule belongs to an IgG class.
42. The Fc fusion protein composition according to claim 30,
wherein the Fc fusion protein composition is Fc-fused fibroblast
growth factor-8.
43. A cell which produces the Fc fusion protein composition
according to claim 30.
45. The cell according to claim 43, which is a mouse myeloma
cell.
46. The cell according to claim 45, wherein the mouse myeloma cell
is NS0 cell or SP2/0-Ag14 cell.
47. The cell according to claim 43, which is selected from the
group consisting of the following (a) to (g): (a) a CHO cell
derived from a Chinese hamster ovary tissue; (b) a rat myeloma cell
line YB2/3HL.P2.G11.16Ag.20 line; (c) a BHK cell derived from a
Syrian hamster kidney tissue; (d) an antibody-producing hybridoma
cell; (e) a human leukemic cell line Namalwa cell; (f) an embryonic
stem cell; (g) a fertilized egg cell.
48. A process for producing an Fc fusion protein
Description
BACKGROUND OF TE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to a method for enhancing a
binding activity of an antibody composition to Fc.gamma. receptor
IIIa, which comprises modifying a complex N-glycoside-linked sugar
chain which is bound to the Fc region of an antibody molecule; a
method for enhancing an antibody-dependent cell-mediated cytotoxic
activity of an antibody composition; a process for producing an
antibody composition having an enhanced binding activity to
Fc.gamma. receptor IIIa; a method for detecting the ratio of a
sugar chain in which fucose is not bound to N-acetylglucosamine in
the reducing end in the sugar chain among total complex
N-glycoside-linked sugar chains bound to the Fc region in an
antibody composition; an Fc fusion protein composition produced by
using a cell resistant to a lectin which recognizes a sugar chain
in which 1-position of fucose is bound to 6-position of
N-acetylglucosamine in the reducing end through .alpha.-bond in a
complex N-glycoside-linked sugar chain; and a process for producing
the same.
[0003] 2. Brief Description of the Background Art
[0004] Since antibodies have high binding activity, binding
specificity and high stability in blood, their applications to
diagnosis, prevention and treatment of various human diseases have
been attempted [Monoclonal Antibodies: Principles and Applications,
Wiley-Liss, Inc., Chapter 2.1 (1995)]. Also, production of a
humanized antibody such as a human chimeric antibody or a human
complementarity determining region (hereinafter referred to as
"CDR")-grafted antibody from a non-human animal antibody have been
attempted by using genetic recombination techniques. The human
chimeric antibody is an antibody in which its antibody variable
region (hereinafter referred to as "V region") is derived from a
non-human animal antibody and its constant region (hereinafter
referred to as "C region") is derived from a human antibody. The
human CDR-grafted antibody is an antibody in which the CDR of a
human antibody is replaced by CDR derived from a non-human animal
antibody.
[0005] It has been revealed that five classes, namely IgM, IgD,
IgG, IgA and IgE, are present in mammal antibodies. Antibodies of
human IgG class are mainly used for the diagnosis, prevention and
treatment of various human diseases because they have functional
characteristics such as long half-life in blood and various
effector functions [Monoclonal Antibodies: Principles and
Applications, Wiley-Liss, Inc., Chapter 1 (1995)]. The human IgG
class antibody is further classified into the following 4
subclasses: IgG1, IgG2, IgG3 and IgG4. A large number of studies
have so far been conducted for antibody-dependent cell-mediated
cytotoxic activity (hereinafter referred to as "ADCC activity") and
complement-dependent cytotoxic activity (hereinafter referred to as
"CDC activity") as effector functions of the IgG class antibody,
and it has been reported that among antibodies of the human IgG
class, the IgG1 subclass has the highest ADCC activity and CDC
activity [Chemical Immunology, 65, 88 (1997)]. In view of the
above, most of the anti-tumor humanized antibodies, including
commercially available Rituxan and Herceptin, which require high
effector functions for the expression of their effects, are
antibodies of the human IgG1 subclass.
[0006] Expression of ADCC activity and CDC activity of the human
IgG1 subclass antibodies requires binding of the Fc region of the
antibody to a receptor for an antibody (hereinafter referred to as
"Fc.gamma.R") existing on the surface of effector cells such as
killer cells, natural killer cells or activated macrophages and
various complement components are bound. Regarding the binding, it
has been suggested that several amino acid residues in the hinge
region and the second domain of C region (hereinafter referred to
as "C.gamma.2 domain") of the antibody are important [Eur. J.
Immunol., 23, 1098 (1993), Immunology, 86, 319 (1995), Chemical
Immunology, 65, 88 (1997)] and that a sugar chain in the C.gamma.2
domain [Chemical Immunology, 65, 88 (1997)] is also important.
[0007] Regarding the sugar chain, Boyd et al. have examined effects
of a sugar chain on the ADCC activity and CDC activity by treating
a human CDR-grafted antibody CAMPATH-1H (human IgG1 subclass)
produced by a Chinese hamster ovary cell (hereinafter referred to
as "CHO cell") or a mouse myeloma NS0 cell (hereinafter referred to
as "NS0 cell") with various glycosidases, and reported that
elimination of sialic acid in the non-reducing end did not have
influence upon both activities, but the CDC activity alone was
affected by further removal of galactose residue and about 50% of
the activity was decreased, and that complete removal of the sugar
chain caused disappearance of both activities [Molecular Immunol.,
32, 1311 (1995)]. Also, Lifely et al have analyzed the sugar chain
bound to a human CDR-grafted antibody CAMPATH-1H (human IgG1
subclass) which was produced by CHO cell, NS0 cell or rat myeloma
Y0 cell (hereinafter referred to as "Y0 cell"), measured its ADCC
activity, and reported that the CAMPATH-1H derived from Y0 cell
showed the highest ADCC activity, suggesting that
N-acetylglucosamine (hereinafter sometimes referred to as "GlcNAc")
at the bisecting position is important for the activity
[Glycobiology, 5, 813 (1995), WO99/54342]. These reports indicate
that the structure of the sugar chain plays an important role in
the effector functions of human antibodies of IgG1 subclass and
that it is possible to prepare an antibody having much higher
effector function by modifying the structure of the sugar chain.
However, structures of sugar chains are various and complex
actually, and it cannot be said that an important structure for the
effector function was completely identified.
[0008] Thus, the sugar chain bound to the CH2 domain of an IgG
class antibody has great influence on the induction of effector
functions of an antibody. As described above, some of effector
functions of an antibody are exerted via interaction with
Fc.gamma.R present on the effector cell surface [Annu. Rev.
Immunol., 18, 709 (2000), Annu. Rev. Immunol., 19, 275 (2001)].
[0009] It has been found that 3 different types are present in
Fc.gamma.R, and they are respectively called Fc.gamma.RI (CD64),
Fc.gamma.RII (CD32) and Fc.gamma.RIII (CD16). In human,
Fc.gamma.RII and Fc.gamma.RIII are further classified into
Fc.gamma.RIIa and Fc.gamma.RIIb, and Fc.gamma.RIIIa and
Fc.gamma.RIIIb, respectively. Fc.gamma.R is a membrane protein
belonging to the immunoglobulin super family, Fc.gamma.RII and
Fc.gamma.RIII have an .alpha. chain having an extracellular region
containing two immunoglobulin-like domains, Fc.gamma.RI has an
.alpha. chain having an extracellular region containing three
immunoglobulin-like domains, as a constituting component, and the
.alpha. chain is involved in the IgG binding activity. In addition,
Fc.gamma.RI and Fc.gamma.RIII have a .gamma. chain or .zeta. chain
as a constituting component which has a signal transduction
function in association with the .alpha. chain [Annu. Rev. Immunol,
18, 709 (2000), Annu. Rev. Immunol, 19, 275 (2001)).
[0010] Fc.gamma.RI is a high affinity receptor having a binding
constant (hereinafter referred to as "K.sub.A") value of 10.sup.8
to 10.sup.9 M.sup.-1 and also has high binding activity for
monomeric IgG [Ann. Hematol., 76, 231 (1998)]. On the other hand,
Fc.gamma.RII and Fc.gamma.RII are low affinity receptors which have
a low K.sub.A value of 10.sup.5 to 10.sup.7 M.sup.-1 for monomeric
IgG and efficiently bind to an IgG immune complex polymerized by
binding to an antigen or the like 8 Ann. Hematol., 76, 231 (1998)].
Based on its functions, Fc.gamma.R is classified into an activating
receptor and an inhibitory receptor [Annu. Rev. Immunol., 19, 275
(2001)].
[0011] In the activating receptor, there is a sequence consisting
of 19 amino acid residues, called immunoreceptor tyrosine-based
activation motif (hereinafter referred to as "ITAM") in the
intracellular region of the .alpha. chain or the associating
.gamma. chain or .zeta. chain. According to the binding of an IgG
immune complex, a tyrosine kinase such as Src or Syk which
interacts with ITAM is activated to induce various activation
reactions.
[0012] In the inhibitory receptor, there is a sequence consisting
of 13 amino acid residues, called immunoreceptor tyrosine-based
inhibitory motif (hereinafter referred to as "ITIM") in the
intracellular region of the CL chain. When ITIM is phosphorylated
via its association with the activating receptor, various reactions
including activation of a phosphatase called SHIP are induced to
suppress activation signal from the activation receptor.
[0013] In human, the high affinity Fc.gamma.RI and the low affinity
Fc.gamma.RIIa and Fc.gamma.RIIIa function as activating receptors.
In Fc.gamma.RI, an ITAM sequence is present in the intracellular
region of the associated .gamma. chain. Fc.gamma.RI is expressed on
macrophages, monocytes, dendritic cells, neutrophils, eosinophils
and the like. Fc.gamma.RIIa comprises a single .alpha. chain, and
an ITAM-like sequence is present in the intracellular region.
Fc.gamma.RIIa is expressed on macrophages, mast cells, monocytes,
dendritic cells, Langerhans cells, neutrophils, eosinophils,
platelets and a part of B cells. Fc.gamma.RIIIa has an ITAM
sequence present in the intracellular region of the associated
.gamma. chain or .zeta. chain and is expressed on NK cells,
macrophages, monocytes, mast cells, dendritic cells, Langerhans
cells, eosinophils and the like, but is not expressed on
neutrophils, B cells and T cells.
[0014] On the other hand, the low affinity receptor Fc.gamma.RIIb
comprises a single .alpha. chain, and the amino acid sequence in
the extracellular region has homology of about 90% with
Fc.gamma.RIIa. However, since an ITMI sequence is present in the
intracellular region, it functions as a suppressing receptor.
Fc.gamma.RIIb is expressed on B cells, macrophages, mast cells,
monocytes, dendritic cells, Langerhans cells, basophils,
neutrophils and eosinophils, but is not expressed on NK cells and T
cells. Fc.gamma.RIIIb comprises a single .alpha. chain, and the
amino acid sequence in the extracellular region has a homology of
about 95% with Fc.gamma.RIIIa. However, it is specifically
expressed on neutrophils as a glycosylphosphatidylinositol
(hereinafter referred to as "GPI")-anchored membrane protein.
Fc.gamma.RIIIb binds to an IgG immune complex but cannot activate
cells by itself, and it is considered to function via its
association with a receptor having an ITAM sequence such as
Fc.gamma.RIIa. Thus, in vivo effector functions of IgG class
antibodies are obtained as the result of complex interaction with
activating and suppressing Fc.gamma.Rs expressed on various
effector cells.
[0015] It is considered that ADCC activity as one of the effector
functions of IgG class antibodies is generated as a result of
activation of effector cells such as NK cells, neutrophils,
monocytes and macrophages, and among these, NK cells play an
important role [Blood, 76, 2421 (1990), Trends in Immunol., 22, 633
(2001), Int. Rev. Immunol., 20, 503 (2001)].
[0016] Fc.gamma.R expressed on NK cells is Fc.gamma.RIIIa.
Accordingly, it is considered that the ADCC activity can be
enhanced by enhancing the activation signal from Fc.gamma.RIIa
expressed on the NK cells.
[0017] As Fc fusion protein, Etanercept (trade name: Enbrel,
manufactured by Immunex) (U.S. Pat. No. 5,605,690) and Alefacept
(trade name: Arnevive, manufactured by Biogen) (U.S. Pat. No.
5,914,111) and the like are known. Also, it is known that it has no
ADCC activity when CH2 domain of an antibody is absent.
SUMMARY OF THE INVENTION
[0018] The present invention relates to the following (1) to
(48).
[0019] (1) A method for enhancing a binding activity of an antibody
composition to Fc.gamma. receptor IIIa, which comprises modifying a
complex N-glycoside-linked sugar chain which is bound to the Fc
region of an antibody molecule.
[0020] (2) The method according to (1), wherein the modification of
a complex N-glycoside-linked sugar chain which is bound to the Fc
region of an antibody molecule is to bind a sugar chain in which
1-position of fucose is not bound to 6-position of
N-acetylglucosamine in the reducing end through .alpha.-bond in the
complex N-glycoside-linked sugar chain to the Fc region in the
antibody molecule.
[0021] (3) The method according to (1) or (2), wherein the sugar
chain is synthesized by a cell in which the activity of a protein
relating to modification of a sugar chain in which fucose is bound
to N-acetylglucosamine in the reducing end in the complex
N-glycoside-linked sugar chain is decreased or deleted.
[0022] (4) The method according to (3), wherein the protein
relating to modification of a sugar chain in which fucose is bound
to N-acetylglucosamine in the reducing end in the complex
NV-glycoside-linked sugar chain is selected from the group
consisting of the following (a), (b) and (c):
[0023] (a) a protein relating to synthesis of an intracellular
sugar nucleotide, GDP-fucose;
[0024] (b) a protein relating to modification of a sugar chain in
which 1-position of fucose is bound to 6-position of
N-acetylglucosamine in the reducing end through .alpha.-bond in a
complex N-glycoside-linked sugar chain;
[0025] (c) a protein relating to transport of an intracellular
sugar nucleotide, GDP-fucose to the Golgi body.
[0026] (5) The method according to (3) or (4), wherein the cell is
resistant to a lectin which recognizes a sugar chain structure in
which 1-position of fucose is bound to 6-position of
N-acetylglucosamine in the reducing end through (.alpha.-bond in a
complex N-glycoside-linked sugar chain.
[0027] (6) The method according to any one of (3) to (5), wherein
the cell is resistant to at least one lectin selected from the
group consisting of the following (a) to (d):
[0028] (a) a Lens culinaris lectin;
[0029] (b) a Pisum sativum lectin;
[0030] (c) a Vicia faba lectin;
[0031] (d) an Aleuria aurantia lectin.
[0032] (7) The method according to any one of (3) to (6), wherein
the cell is selected from the group consisting of a yeast, an
animal cell, an insect cell and a plant cell.
[0033] (8) The method according to any one of (3) to (7), wherein
the cell is selected from the group consisting of the following (a)
to (i):
[0034] (a) a CHO cell derived from a Chinese hamster ovary
tissue;
[0035] (b) a rat myeloma cell line YB2/3HL.P2.G11.16Ag.20 cell;
[0036] (c) a BHK cell derived from a Syrian hamster kidney
tissue;
[0037] (d) a mouse myeloma cell line NS0 cell;
[0038] (e) a mouse myeloma cell line SP2/0-Ag14 cell;
[0039] (f) a hybridoma cell;
[0040] (g) a human leukemic cell line Namalwa cell;
[0041] (h) an embryonic stem cell;
[0042] (i) a fertilized egg cell.
[0043] (9) The method according to any one of (1) to (8), wherein
the antibody molecule is selected from the group consisting of the
following (a) to (d):
[0044] (a) a human antibody;
[0045] (b) a humanized antibody;
[0046] (c) an antibody fragment comprising the Fc region of (a) or
(b);
[0047] (d) a fusion protein comprising the Fc region of (a) or
(b).
[0048] (10) The method according to any one of (1) to (9), wherein
the antibody molecule belongs to an IgG class.
[0049] (11) The method according to any one of (1) to (10),
wherein, in the complex N-glycoside-linked sugar chain which is
bound to the Fc region of an antibody molecule, the ratio of a
sugar chain in which 1-position of fucose is not bound to
6-position of N-acetylglucosamine in the reducing end through
.alpha.-bond is 20% or more of total complex N-glycoside-linked
sugar chains.
[0050] (12) A method for enhancing an antibody-dependent
cell-mediated cytotoxic activity of an antibody composition, which
comprises using the method according to any one of (1) to (11).
[0051] (13) A process for producing an antibody composition having
an enhanced binding activity to Fc.gamma. receptor IIIa, which
comprises modifying a complex N-glycoside-linked sugar chain which
is bound to the Fc region of an antibody molecule.
[0052] (14) The process according to (13), wherein the modification
of a complex N-glycoside-linked sugar chain which is bound to the
Fc region of an antibody molecule is to bin a sugar chain in which
1-position of fucose is not bound to 6-position of
N-acetylglucosamine in the reducing end through .alpha.-bond in the
complex N-glycoside-linked sugar chain to the Fc region in the
antibody molecule.
[0053] (15) The process according to (13) or (14), wherein the
sugar chain is synthesized by a cell in which the activity of a
protein relating to modification of a sugar chain in which fucose
is bound to N-acetylglucosamine in the reducing end in the complex
N-glycoside-linked sugar chain is decreased or deleted.
[0054] (16) The process according to (15), wherein the protein
relating to modification of a sugar chain in which fucose is bound
to N-acetylglucosamine in the reducing end in the complex
N-glycoside-linked sugar chain is selected from the group
consisting of the following (a), (b) and (c):
[0055] (a) a protein relating to synthesis of an intracellular
sugar nucleotide, GDP-fucose;
[0056] (b) a protein relating to modification of a sugar chain in
which 1-position of fucose is bound to 6-position of
N-acetylglucosamine in the reducing end through .alpha.-bond in a
complex N-glycoside-linked sugar chain;
[0057] (c) a protein relating to transport of an intracellular
sugar nucleotide, GDP-fucose to the Golgi body.
[0058] (17) The process according to (15) or (16), wherein the cell
is resistant to a lectin which recognizes a sugar chain structure
in which 1-position of fucose is bound to 6-position of
NV-acetylglucosamine in the reducing end through or .alpha.-bond in
a complex N-glycoside-linked sugar chain.
[0059] (18) The process according to any one of (15) to (17),
wherein the cell is resistant to at least one lectin selected from
the group consisting of the following (a) to (d):
[0060] (a) a Lens culinaris lectin;
[0061] (b) a Pisum sativum lectin;
[0062] (c) a Vicia faba lectin;
[0063] (d) an Aleuria aurantia lectin.
[0064] (19) The process according to any one of (15) to (18),
wherein the cell is selected from the group consisting of a yeast,
an animal cell, an insect cell and a plant cell.
[0065] (20) The process according to any one of (15) to (19),
wherein the cell is selected from the group consisting of the
following (a) to (i):
[0066] (a) a CHO cell derived from a Chinese hamster ovary
tissue;
[0067] (b) a rat myeloma cell line YB2/3HL.P2.G11.16Ag.20 cell;
[0068] (c) a BHK cell derived from a Syrian hamster kidney
tissue;
[0069] (d) a mouse myeloma cell line NS0 cell;
[0070] (e) a mouse myeloma cell line SP2/0-Ag14 cell;
[0071] (f) a hybridoma cell;
[0072] (g) a human leukemic cell line Namalwa cell;
[0073] (h) an embryonic stem cell;
[0074] (i) a fertilized egg cell.
[0075] (21) The process according to any one of (13) to (20),
wherein the antibody molecule is selected from the group consisting
of the following (a) to (d):
[0076] (a) a human antibody;
[0077] (b) a humanized antibody;
[0078] (c) an antibody fragment comprising the Fc region of (a) or
(b);
[0079] (d) a fusion protein comprising the Fc region of (a) or
(b).
[0080] (22) The process according to any one of (13) to (21),
wherein the antibody molecule belongs to an IgG class.
[0081] (23) The method according to any one of (13) to (22),
wherein, in the complex N-glycoside-linked sugar chain which is
bound to the Fc region of an antibody molecule, the ratio of a
sugar chain in which 1-position of fucose is not bound to
6-position of N-acetylglucosamine in the reducing end through
.alpha.-bond is 20% or more of total complex N-glycoside-linked
sugar chains.
[0082] (24) A process for producing an antibody composition having
an increased antibody-dependent cell-mediated cytotoxic activity,
which comprises using the process according to (12).
[0083] (25) An antibody composition produced by the process
according to any one of (13) to (24).
[0084] (26) A method for detecting the ratio of a sugar chain in
which fucose is not bound to N-acetylglucosamine in the reducing
end in the sugar chain among total complex N-glycoside-linked sugar
chains bound to the Fc region in an antibody composition, which
comprises; reacting an antigen with a tested antibody composition
to form a complex of the antigen and the antibody composition,
contacting the complex with an Fc.gamma. receptor IIIa to measure
the binding activity to the Fc.gamma. receptor IIIa; and detecting
the ratio of a sugar chain in a standard antibody composition with
a standard curve showing the binding activity to the Fc.gamma.
receptor IIIa.
[0085] (27) A method for detecting the antibody-dependent
cell-mediated cytotoxic activity in an antibody composition, which
comprises: reacting an antigen with a tested antibody composition
to form a complex of the antigen and the antibody composition;
contacting the complex with an Fc.gamma. receptor IIIa to measure
the binding activity to the Fc.gamma. receptor IIIa; and detecting
the ratio of a sugar chain in a standard antibody composition with
a standard curve showing the binding activity to the Fc.gamma.
receptor IIIa.
[0086] (28) A method for detecting the ratio of a sugar chain in
which fucose is not bound to N-acetylglucosamine in the reducing
end in the sugar chain among total complex N-glycoside-linked sugar
chains bound to the Fc region in an antibody composition, which
comprises: contacting a tested antibody composition with a
Fc.gamma. receptor IIIa to measure the binding activity of the
antibody composition to the Fc.gamma. receptor IIIa; and detecting
the ratio of a sugar chain in a standard antibody composition with
a standard curve showing the binding activity to the Fc.gamma.
receptor IIIa.
[0087] (29) A method for detecting the antibody-dependent
cell-mediated cytotoxic activity in an antibody composition, which
comprises: contacting a tested antibody composition with a
Fc.gamma. receptor IIIa to measure the binding activity of the
antibody composition to the Fc.gamma. receptor IIIa, and detecting
the ratio of a sugar chain in a standard antibody composition with
a standard curve showing the binding activity to the Fc.gamma.
receptor IIIa.
[0088] (30) An Fc fusion protein composition produced by using a
cell resistant to a lectin which recognizes a sugar chain structure
in which 1-position of fucose is bound to 6-position of
N-acetylglucosamine in the reducing end through .alpha.-bond in a
complex N-glycoside-linked sugar chain.
[0089] (31) The Fc fusion protein composition according to (30),
wherein the cell is selected from the group consisting of the
following (a), (b) and (c):
[0090] (a) an enzyme protein relating to synthesis of an
intracellular sugar nucleotide, GDP-fucose;
[0091] (b) an enzyme protein relating to modification of a sugar
chain in which 1-position of fucose is bound to 6-position of
N-acetylglucosamine in the reducing end through .alpha.-bond in a
complex N-glycoside-linked sugar chain;
[0092] (c) a protein relating to transport of an intracellular
sugar nucleotide, GDP-fucose to the Golgi body,
[0093] wherein the activity of the protein is decreased or
deleted.
[0094] (32) The Fc fusion protein composition according to (30) or
(31), wherein the cell is resistant to at least one lectin selected
from the group consisting of the following (a) to (d):
[0095] (a) a Lens culinaris lectin;
[0096] (b) a Pisum sativum lectin;
[0097] (c) a Vicia faba lectin;
[0098] (d) an Aleuria aurantia lectin.
[0099] (33) The Fc fusion protein composition according to any one
of (30) to (32), wherein the cell is a cell into which a gene
encoding an Fc fusion protein is introduced.
[0100] (34) The Fc fusion protein composition according to (33),
wherein the Fc is derived from an IgG class of an antibody
molecule.
[0101] (35) The Fc fusion protein composition according to any one
of (30) to (34), wherein the cell is selected from the group
consisting of a yeast, an animal cell, an insect cell and a plant
cell.
[0102] (36) The Fc fusion protein composition according to any one
of (30) to (35), wherein the cell is a mouse myeloma cell.
[0103] (37) The Fc fusion protein composition according to (36),
wherein the mouse myeloma cell is NS0 cell or SP2/0-Ag14 cell.
[0104] (38) The Fc fusion protein composition according to any one
of (30) to (37), wherein the cell is selected from the group
consisting of the following (a) to (g):
[0105] (a) a CHO cell derived from a Chinese hamster ovary
tissue;
[0106] (b) a rat myeloma cell line YB2/3HL.P2.G11.16Ag.20 line;
[0107] (c) a BHK cell derived from a Syrian hamster kidney
tissue;
[0108] (d) an antibody-producing hybridoma cell;
[0109] (e) a human leukemic cell line Namalwa cell;
[0110] (f) an embryonic stem cell;
[0111] (g) a fertilized egg cell.
[0112] (39) An Fc fusion protein composition comprising an Fc
fusion protein having an complex N-glycoside-linked sugar chain at
the Fc region of an antibody molecule, wherein the ratio of a sugar
chain in which fucose is not bound to N-acetylglucosamine in the
reducing end in the sugar chain is 20% or more of total complex
N-glycoside-linked sugar chains which are bound to the Fc region in
the composition.
[0113] (40) The Fc fusion protein composition according to (39),
wherein the sugar chain in which fucose is not bound is a sugar
chain in which 1-position of fucose is not bound to 6-position of
V-acetylglucosamine in the reducing end in the complex
N-glycoside-linked sugar chain through .alpha.-bond.
[0114] (41) The Fc fusion protein composition according to (39) or
(40), wherein the antibody molecule belongs to an IgG class.
[0115] (42) The Fc fusion protein composition according to any one
of (30) to (41), wherein the Fc fusion protein composition is
Fc-fused fibroblast growth factor-8.
[0116] (43) A cell which produces the Fc fusion protein composition
according to any one of (30) to (42).
[0117] (44) The cell according to (43), which is selected from the
group consisting of a yeast, an animal cell, an insect cell and a
plant cell.
[0118] (45) The cell according to (43) or (44), which is a mouse
myeloma cell.
[0119] (46) The cell according to (45), wherein the mouse myeloma
cell is NS0 cell or SP2/0-Ag14 cell.
[0120] (47) The cell according to any one of (43) to (46), which is
selected from the group consisting of the following (a) to (g):
[0121] (a) a CHO cell derived from a Chinese hamster ovary
tissue;
[0122] (b) a rat myeloma cell line YB2/3HL.P2.G11.16Ag.20 line;
[0123] (c) a BHK cell derived from a Syrian hamster kidney
tissue;
[0124] (d) an antibody-producing hybridoma cell;
[0125] (e) a human leukemic cell line Namalwa cell;
[0126] (f) an embryonic stem cell;
[0127] (g) a fertilized egg cell.
[0128] (48) A process for producing an Fc fusion protein
composition, which comprises culturing the cell according to any
one of (43) to (47) in a medium to form and accumulate an Fc fusion
protein composition in the culture, and recovering the Fc fusion
protein composition from the culture.
BRIEF DESCRIPTION OF THE DRAWINGS
[0129] FIG. 1 shows photographs of electrophoresis patterns of
SDS-PAGE of five kinds of purified anti-GD3 chimeric antibodies
(using gradient gel from 4 to 15%). FIG. 1A and FIG. 1B show
results of the electrophoresis under non-reducing conditions and
under reducing conditions, respectively. Lanes 1 to 7 show
electrophoresis patterns of high molecular weight markers,
YB2/0-GD3 chimeric antibody, CHO/DG44-GD3 chimeric antibody,
SP2/0-GD3 chimeric antibody, NS0-GD3 chimeric antibody (302),
NS0-GD3 chimeric antibody (GIT) and low molecular weight markers,
respectively.
[0130] FIG. 2 shows binding activities of five kinds of purified
anti-GD3 chimeric antibodies to GD3, measured by changing the
antibody concentration. The ordinate and the abscissa show the
binding activity to GD3 and the antibody concentration,
respectively. ".largecircle.", ".circle-solid.", ".quadrature.",
".box-solid." and ".DELTA." show the activities of YB2/0-GD3
chimeric antibody, CHO/DG44-GD3 chimeric antibody, SP2/0-GD3
chimeric antibody, NS0-GD3 chimeric antibody (302) and NS0-GD3
chimeric antibody (GIT), respectively.
[0131] FIG. 3 shows ADCC activities of five kinds of purified
anti-GD3 chimeric antibodies to a human melanoma cell line G-361.
The ordinate and the abscissa show the cytotoxic activity and the
antibody concentration, respectively. ".largecircle.",
".circle-solid.", ".quadrature.", ".box-solid." and ".DELTA." show
the activities of YB2/0-GD3 chimeric antibody, CHO/DG44-GD3
chimeric antibody, SP2/0-GD3 chimeric antibody, NS0-GD3 chimeric
antibody (302) and NS0-GD3 chimeric antibody (GIT),
respectively.
[0132] FIG. 4 shows elution patterns of PA-treated sugar chains
prepared from the anti-GD3 chimeric antibody of lot 2, obtained by
analyzing them with reverse phase HPLC. The ordinate and the
abscissa show the relative fluorescence intensity and the elution
time, respectively.
[0133] FIG. 5 shows binding activities of six kinds of anti-GD3
chimeric antibodies having a different ratio of a sugar chain in
which 1-position of fucose is not bound to 6-position of
N-acetylglucosaminethe in the reducing end through .alpha.-bond to
GD3, measured by changing the antibody concentration. The ordinate
and the abscissa show the binding activity to GD3 and the antibody
concentration, respectively. ".circle-solid.", ".quadrature.",
".box-solid.", ".DELTA.", ".tangle-solidup." and "x" show the
activities of anti-GD3 chimeric antibody (50%), anti-GD3 chimeric
antibody (45%), anti-GD3 chimeric antibody (29%), anti-GD3 chimeric
antibody (24%), anti-GD3 chimeric antibody (13%) and anti-GD3
chimeric antibody (7%), respectively.
[0134] FIG. 6 shows results of ADCC activities using an effector
cell of each doner. FIG. 6A and FIG. 6B show ADCC activities of six
kinds of anti-GD3 chimeric antibodies having a different ratio of a
sugar chain in which 1-position of fucose is not bound to
6-position of N-acetylglucosaminethe in the reducing end through
.alpha.-bond against a human melanoma cell line G-361, using
effector cells of the donor A and the doner B, respectively. The
ordinate and the abscissa show the cytotoxic activity and the
antibody concentration, respectively. ".circle-solid.",
".quadrature.", ".box-solid.", ".DELTA.", ".tangle-solidup." and
"x" show the activities of anti-GD3 chimeric antibody (50%),
anti-GD3 chimeric antibody (45%), anti-GD3 chimeric antibody (29%),
anti-GD3 chimeric antibody (24%), anti-GD3 chimeric antibody (13%)
and anti-GD3 chimeric antibody (7%), respectively.
[0135] FIG. 7 shows the relationship between sugar chain contents
in which 1-fucose is not bound to N-acetylglucosamine at the
reduced end in the doner A and the doner B and the ADCC
activities.
[0136] FIG. 8 shows elution patterns of PA-treated sugar chains
prepared from six kinds of anti-GD3 chimeric antibodies, obtained
by analyzing them with reverse phase HPLC. The ordinate and the
abscissa show the relative fluorescence intensity and the elution
time, respectively.
[0137] FIG. 9 shows binding activities of six kinds of anti-CCR4
chimeric antibodies having a different ratio of a sugar chain in
which 1-position of fucose is not bound to 6-position of
N-acetylglucosaminethe in the reducing end through .alpha.-bond
measured by changing the antibody concentration to CCR4. The
ordinate and the abscissa show the binding activity to CCR4 and the
antibody concentration, respectively. ".box-solid.",
".quadrature.", ".tangle-solidup.", ".DELTA.", ".circle-solid." and
".largecircle." show the activities of anti-CCR4 chimeric antibody
(46%), anti-CCR4 chimeric antibody (39%), anti-CCR4 chimeric
antibody (27%), anti-CCR4 chimeric antibody (18%), anti-CCR4
chimeric antibody (9%) and anti-CCR4 chimeric antibody (8%),
respectively.
[0138] FIG. 10 shows ADCC activities of anti-CCR4 chimeric
antibodies having a different ratio of a sugar chain in which
1-position of fucose is not bound to 6-position of
N-acetylglucosaminethe in the reducing end through .alpha.-bond
against CCR4/EL-4 cell, using an effector cell of the donor A. As
effector cells, the effector cells derived from donor A were used.
The ordinate and the abscissa show the cytotoxic activity and the
antibody concentration, respectively. ".box-solid.",
".quadrature.", ".tangle-solidup.", ".DELTA.", ".circle-solid." and
".largecircle." show the activities of anti-CCR4 chimeric antibody
(46%), anti-CCR4 chimeric antibody (39%), anti-CCR4 chimeric
antibody (27%), anti-CCR4 chimeric antibody (18%), anti-CCR4
chimeric antibody (9%) and anti-CCR4 chimeric antibody (8%),
respectively.
[0139] FIG. 11 shows ADCC activities of anti-CCR4 chimeric
antibodies having a different ratio of a sugar chain in which
1-position of fucose is not bound to 6-position
N-acetylglucosaminethe in the reducing end through .alpha.-bond
against CCR4/EL-4 cell, using an effector cell of the donor B. As
effector cells, the effector cells derived from donor B were used.
The ordinate and the abscissa show the cytotoxic activity and the
antibody concentration, respectively. ".box-solid.",
".quadrature.", ".tangle-solidup.", ".DELTA.", ".circle-solid." and
".largecircle." show the activities of anti-CCR4 chim eric antibody
(46%), anti-CCR4 chimeric antibody (39%), anti-CCR4 chimeric
antibody (27%), anti-CCR4 chimeric antibody (18%), anti-CCR4
chimeric antibody (9%) and anti-CCR4 chimeric antibody (8%),
respectively.
[0140] FIG. 12 shows construction of plasmids CHFT8-pCR2.1 and
YBFT8-pCR2.1.
[0141] FIG. 13 shows construction of plasmids CHAc-pBS and
YBAc-pBS.
[0142] FIG. 14 shows construction of plasmids CHFT8d-pCR2.1 and
YBFT8d-pCR2.1.
[0143] FIG. 15 shows construction of plasmids CHAcd-pBS and
YBAcd-pBS.
[0144] FIG. 16 shows results of determination of an
.alpha.1,6-fucosyltransferase (FUT8) transcription product in each
host cell line using competitive RT-PCR. Amounts of the FUT8
transcription product in each host cell line when rat FUT8 sequence
was used as the standard and internal control are shown.
".box-solid." and ".quadrature." show results when CHO cell line
and YB2/0 cell line, respectively, were used as the host cell.
[0145] FIG. 17 shows results of evaluation of ADCC activities of
anti-CCR4 human chimeric antibodies produced by lectin-resistant
cell lines. The ordinate and the abscissa show the cytotoxic
activity and the antibody concentration, respectively.
".quadrature.", ".box-solid.", ".diamond-solid." and
".tangle-solidup." show the activities of antibodies produced by
the clone 5-03, the clone CHO/CCR4-LCA, the clone CHO/CCR4-AAL and
the clone CHO/CCR4-PHA, respectively.
[0146] FIG. 18 shows results of evaluation of ADCC activities of
anti-CCR4 human chimeric antibodies produced by lectin-resistant
clones. The ordinate and the abscissa show the cytotoxic activity
and the antibody concentration, respectively. ".quadrature."
".DELTA." and ".circle-solid." show activities of antibodies
produced by the clone YB2/0 (KM2760#58-35-16), the clone 5-03 and
the clone CHO/CCR4-LCA, respectively.
[0147] FIG. 19 shows elution patterns of PA-treated sugar chains
prepared from purified anti-CCR4 human chimeric antibodies,
obtained by analyzing them with reverse phase HPLC. The ordinate
and the abscissa show the relative fluorescence intensity and the
elution time, respectively. FIG. 27A, FIG. 27B, FIG. 27C and FIG.
27D show results of analyses of antibodies produced by the clone
5-03, the clone CHO/CCR4-LCA, the clone CHO/CCR4-AAL and the clone
CHO/CCR4-PHA, respectively.
[0148] FIG. 20 shows the 1st step of construction of an expression
vector of CHO cell-derived GMD (6 steps in total).
[0149] FIG. 21 shows the 2nd step of construction of the expression
vector of CHO cell-derived GMD (6 steps in total).
[0150] FIG. 22 shows the 3rd step of construction of the expression
vector of CHO cell-derived GMD (6 steps in total).
[0151] FIG. 23 shows the 4th step of construction of the expression
vector of CHO cell-derived GM (6 steps in total).
[0152] FIG. 24 shows the 5th step of construction of the expression
vector of CHO cell-derived GMD (6 steps in total).
[0153] FIG. 25 shows the 6th step of construction of the expression
vector of CHO cell-derived GMD (6 steps in total).
[0154] FIG. 26 shows resistance of GMD-expressed clone CHO/CCR4-LCA
for LCA lectin. The measurement was carried out twice by defining
the survival rate of a group of cells cultured without adding LCA
lectin as 100%. In the drawing, "249" shows the survival rate of
the clone CHO/CCR4-LCA introduced with an expression vector to LCA
lectin. GMD shows resistance of the clone CHO/CCR4-LCA introduced
with a GMD expression vector pAGE249GMD to LCA lectin.
[0155] FIG. 27 shows ADCC activities of an anti-CCR4 chimeric
antibody produced by cells of GMD-expressed clone CHO/CCR4-LCA. The
ordinate and the abscissa show the cytotoxic activity and the
antibody concentration, respectively.
[0156] FIG. 28 shows elution patterns of PA-treated sugar chains
prepared from an anti-CCR4 human chimeric antibody purified from
GMD gene-expressed clone CHO/CCR4-LCA, obtained by analyzing them
with reverse phase HPLC. The ordinate and the abscissa show the
relative fluorescence intensity and the elution time,
respectively.
[0157] FIG. 29 shows a photograph of SDS-PAGE (using 4 to 15%
gradient gel) electrophoresis pattern of purified shFc.gamma.RIIIa
under reduced conditions. Lane 1 and lane M show electrophoresis
patterns of shFc.gamma.RIIIa and molecular weight markers,
respectively.
[0158] FIG. 30 shows binding activities of various anti-GD3
chimeric antibodies to shFc.gamma.RIIIa. The ordinate and abscissa
show the cytotoxic activity and the antibody concentration,
respectively. ".largecircle." and ".circle-solid." show the
activities of anti-GD3 chimeric antibody (45%) and anti-GD3
chimeric antibody (7%), respectively.
[0159] FIG. 31 shows binding activities of various anti-fibroblast
growth factor-8 (FGF-8) chimeric antibodies to shFc.gamma.RIIIa.
The ordinate and abscissa show the cytotoxic activity and the
antibody concentration, respectively. ".largecircle." and
".circle-solid." show the activities of anti-FGF-8 chimeric
antibody (58%) and anti-FGF-8 chimeric antibody (13%),
respectively.
[0160] FIG. 32 shows binding activities of various anti-CCR4
chimeric antibodies to shFc.gamma.RIIa. In FIG. 32A, the ordinate
and abscissa show the cytotoxic activity and the antibody
concentration, respectively. ".largecircle.", ".circle-solid.",
".quadrature.", ".box-solid.", ".DELTA.", ".tangle-solidup." and
"x" show the activities of anti-CCR4 chimeric antibody (87%),
anti-CCR4 chimeric antibody (46%), anti-CCR4 chimeric antibody
(39%), anti-CCR4 chimeric antibody (27%), anti-CCR4 chimeric
antibody (18%), anti-CCR4 chimeric antibody (9%) and anti-CCR4
chimeric antibody (8%), respectively. In FIG. 32B, the ordinate and
abscissa show the cytotoxic activity and the ratio of a sugar chain
in which 1-position of fucose is not bound to 6-position of
N-acetylglucosamine in the reducing end through .alpha.-bond,
respectively. ".circle-solid." and ".largecircle." show the
activities at 40 .mu.g/mL and 4 .mu.g/mL, respectively.
[0161] FIG. 33 shows binding activities of various anti-CCR4
chimeric antibodies to shFc.gamma.RIIIa. The ordinate and abscissa
show the cytotoxic activity and the antibody concentration,
respectively. ".largecircle.", ".DELTA." and ".circle-solid." show
the activities of anti-CCR4 chimeric antibody (87%), anti-CCR4
chimeric antibody (48%) and anti-CCR4 chimeric antibody (8%),
respectively.
[0162] FIG. 34 shows ADCC activities of various anti-GD3 chimeric
antibodies to human myeloma cell line G-361. The ordinate and
abscissa show the cytotoxic activity and the antibody
concentration, respectively. ".tangle-solidup." and
".circle-solid." show the activities of anti-GD3 chimeric antibody
(42%) and anti-GD3 chimeric antibody (7%), respectively.
[0163] FIG. 35 shows results of measurement of binding activities
of FGF-8/Fc fusion proteins to KM1334. The ordinate and abscissa
show the binding activity and the antibody concentration,
respectively. ".box-solid." and ".largecircle." show the activities
of FGF-8/Fc fusion proteins produced by the cell line YB2/0 and the
cell line CHO/DG44, respectively, as the host cell.
[0164] FIG. 36 shows results of measurement of binding activities
of various FGF-8/Fc fusion proteins to shFc.gamma.RIIIa(V). The
ordinate and abscissa show the binding activity and the antibody
concentration, respectively. ".box-solid." and ".largecircle." show
the activities of FGF8/Fc fusion proteins produced by the cell line
YB2/0 and the cell line CHO/DG44, respectively, as the host
cell.
[0165] FIG. 37 show a construction step of a plasmid CHO-GMD in
which the 5'-terminal of a clone 34-2 is introduced into the
5'-terminal of a CHO cell-derived GMD cDNA clone 22-8.
[0166] FIG. 38 shows construction steps of plasmid pKANTEX1334H and
pKANTEX 1334.
DETAILED DESCRIPTION OF THE INVENTION
[0167] The present invention relates to a method for enhancing a
binding activity of an antibody composition to Fc.gamma. receptor
IIIa, which comprises modifying a complex N-glycoside-linked sugar
chain which is bound to the Fc region of an antibody molecule.
[0168] In the present invention, the antibody molecule may be any
antibody molecule, so long as it is a molecule comprising the Fc
region of an antibody. Examples include an antibody, an antibody
fragment, a fusion protein comprising an Fc region, and the
like.
[0169] An antibody is a protein, which is produced in vivo by
immunization as the result of extra-antigen stimulation. The
antibody has a specific binding activity to an antigen. The
antibody includes an antibody secreted by a hybridoma cell prepared
from a spleen cell of an animal immunized with an antigen, an
antibody prepared by genetic engineering technique, i.e., an
antibody obtained by introducing an antibody gene-inserted antibody
expression vector into a host cell; and the like. Examples include
an antibody produced by a hybridoma, a humanized antibody, a human
antibody and the like.
[0170] A hybridoma is a cell which is obtained by cell fusion
between a B cell obtained by immunizing a non-human mammal with an
antigen and a myeloma cell derived from mouse or the like and can
produce a monoclonal antibody having the desired antigen
specificity.
[0171] The humanized antibody includes a human chimeric antibody, a
human CDR-grafted antibody and the like.
[0172] A human chimeric antibody is an antibody which comprises an
antibody heavy chain V region (hereinafter referred to heavy chain
as "H chain", and referred to as "HV" or "VH") and an antibody
light chain V region (hereinafter referred to light chain as "L
chain", and referred to as "LV" or "VL"), both of a non-human
animal, a human antibody H chain C region (hereinafter also
referred to as "CH") and a human antibody L chain C region
(hereinafter also referred to as "CL"). The non-human animal may be
any animal such as mouse, rat, hamster or rabbit, so long as a
hybridoma can be prepared therefrom.
[0173] The human chimeric antibody can be produced by obtaining
cDNAs encoding VH and VL from a monoclonal antibody-producing
hybridoma, inserting them into an expression vector for host cell
having genes encoding human antibody CH and human antibody CL to
thereby construct a vector for expression of human chimeric
antibody, and then introducing the vector into a host cell to
express the antibody.
[0174] The CH of human chimeric antibody may be any CH, so long as
it belongs to human immunoglobulin (hereinafter referred to as
"hIg") can be used. Those belonging to the hIgG class are preferred
and any one of the subclasses belonging to the hIgG class such as
hIgG1, hIgG2, hIgG3 and hIgG4 can be used. Also, as the CL of human
chimeric antibody, any CL can be used, so long as it belongs to the
hIg class, and those belonging to the .kappa. class or .lambda.
class can also be used.
[0175] A human CDR-grafted antibody is an antibody in which amino
acid sequences of CDRs of VH and VL of a non-human animal antibody
are grafted into appropriate positions of VH and VL of a human
antibody.
[0176] The human CDR-grafted antibody can be produced by
constructing cDNAs encoding V regions in which CDR sequences of VH
and VL of a non-human animal antibody are grafted into CDR
sequences of VH and VL of a desired human antibody, inserting them
into an expression vector for host cell having genes encoding human
antibody CH and human antibody CL to thereby construct a vector for
expression of human CDR-grafted antibody, and then introducing the
expression vector into a host cell to express the human CDR-grafted
antibody.
[0177] The CH of human CDR-grafted antibody may be any CH, so long
as it belongs to the hIg. Those of the hIgG class are preferred and
any one of the subclasses belonging to the hIgG class, such as
hIgG1, hIgG2, hIgG3 and hIgG4, can be used. Also, as the CL of
human CDR-grafted antibody, any CL can be used, so long as it
belongs to the hIg class, and those belonging to the .kappa. class
or .lambda. class can also be used.
[0178] A human antibody is originally an antibody naturally
existing in the human body, but it also includes antibodies
obtained from a human antibody phage library, a human
antibody-producing transgenic non-human animal and a human
antibody-producing transgenic plant, which are prepared based on
the recent advance in genetic engineering, cell engineering and
developmental engineering techniques.
[0179] Regarding the antibody existing in the human body, a
lymphocyte capable of producing the antibody can be cultured by
isolating a human peripheral blood lymphocyte, immortalizing it by
its infection with EB virus or the like and then cloning it, and
the antibody can be purified from the culture.
[0180] The human antibody phage library is a library in which
antibody fragments such as Fab and single chain antibody are
expressed on the phage surface by inserting a gene encoding an
antibody prepared from a human B cell into a phage gene. A phage
expressing an antibody fragment having the desired antigen binding
activity can be recovered from the library based on the activity to
bind to an antigen-immobilized substrate. The antibody fragment can
be converted further into a human antibody molecule comprising two
full H chains and two full L chains by genetic engineering
techniques.
[0181] A human antibody-producing transgenic non-human animal is an
animal in which a human antibody gene is introduced into cells.
Specifically, a human antibody-producing transgenic mouse can be
prepared by introducing a human antibody gene into ES cell of a
mouse, transplanting the ES cell into an early stage embryo of
other mouse and then developing it. By introducing a human
antibody-gene into a fertilized egg of an animal and developing it,
the transgenic non-human animal can be also prepared. Regarding the
preparation method of a human antibody from the human
antibody-producing transgenic non-human animal, the human antibody
can be produced and accumulated in a culture by obtaining a human
antibody-producing hybridoma by a hybridoma preparation method
usually carried out in non-human mammals and then culturing it.
[0182] The transgenic non-human animal includes cattle, sheep,
goat, pig, horse, mouse, rat, fowl, monkey, rabbit and the
like.
[0183] Moreover, in the present invention, the antibody is
preferably an antibody which recognizes a tumor-related antigen, an
antibody which recognizes an allergy- or inflammation-related
antigen, an antibody which recognizes cardiovascular
disease-related antigen, an antibody which recognizes autoimmune
disease-related antigen or an antibody which recognizes a viral or
bacterial infection-related antigen. Also, the class of the
antibody is preferably IgG.
[0184] An antibody fragment is a fragment which comprises at least
part of the Fc region of the above antibody. The Fc region is a
region at the C-terminal side of H chain of an antibody, such as
CH2 region and CH3 region, and includes a natural type and a mutant
type. The at least part of the Fc region is preferably a fragment
comprising CH2 region, and more preferably a region comprising
aspartic acid at position 1 existing in CH2 region. The Fc region
of the IgG class is from Cys at position 226 to the C-terminal or
from Pro at position 230 to the C-terminal according to the
numbering of EU Index of Kabat et al. [Sequences of Proteins of
Immunological Interest, 5.sup.th Ed., Public Health Service,
National Institutes of Health, Bethesda, Md. (1991)]. The antibody
fragment includes an H chain monomer, an H chain dimer and the
like.
[0185] A fusion protein comprising a part of Fc region is a
substance in which an antibody comprising the part of Fc region of
an antibody or the antibody fragment is fused with a protein such
as an enzyme or a cytokine (hereinafter referred to as "Fc fusion
protein").
[0186] In the present invention, N-glycoside-linked sugar chain
bound to the Fc region of the antibody molecule includes a complex
type in which the non-reducing end side of the core structure has
one or more parallel branches of galactose-N-acetylglucosamine
(hereinafter referred to as "Gal-GlcNAc") and further the
non-reducing end side of Gal-GlcNAc has a structure of sialic acid,
bisecting N-acetylglucosamine or the like.
[0187] Since the Fc region in the antibody molecule has positions
to which N-glycoside-linked sugar chains are separately bound, two
sugar chains are bound per one antibody molecule. Since many sugar
chains having different structures are present for the two
N-glycoside-linked sugar chains bound to the antibody, homology of
antibody molecules can be judged in view of the sugar chain
structure bound to the Fc region.
[0188] The antibody composition is a composition which comprises an
antibody molecule having complex N-glycoside-linked sugar chains in
the Fc region, and may comprise an antibody molecule having the
same sugar chain structure or, an antibody molecule having
different sugar chain structures.
[0189] Modification of the N-glycoside-linked sugar chain bound to
the Fc region of an antibody molecule is preferably carried out by
binding a sugar chain in which fucose is not bound to
N-acetylglucosamine in the reducing end in the complex
N-glycoside-linked sugar chain to the Fc region of an antibody
molecule.
[0190] In the present invention, the sugar chain in which fucose is
not bound to N-acetylglucosamine in the reducing end in the complex
N-glycoside-linked sugar chain is a complex M-glycoside-linked
sugar chain in which 1-position of fucose is not bound to
6-position of N-acetylglucosamine in the reducing end through
.alpha.-bond in the complex N-glycoside-linked sugar chain.
[0191] The sugar chain can be synthesized by a cell in which the
activity of an enzyme protein relating to the modification of a
sugar chain in which fucose is not bound to N-acetylglucosamine in
the reducing end in the complex N-glycoside-linked sugar chain is
decreased or deleted.
[0192] In the present invention, the enzyme protein relating to the
modification of a sugar chain in which 1-position of fucose is not
bound to 6-position of N-acetylglucosamine in the reducing end in
the complex N-glycoside-linked sugar chain includes:
[0193] (a) an enzyme protein relating to synthesis of an
intracellular sugar nucleotide, GDP-fucose (hereinafter referred to
as "GDP-fucose synthase");
[0194] (b) an enzyme protein relating to modification of a sugar
chain in which 1-position of fucose is bound to 6-position of
N-acetylglucosamine in the reducing end through .alpha.-bond in a
complex N-glycoside-linked sugar chain (hereinafter referred to as
".alpha.1,6-fucose modifying enzyme"); and
[0195] (c) a protein relating to transport of an intracellular
sugar nucleotide, GDP-fucose, to the Golgi body (hereinafter
referred to as "GDP-fucose transport protein).
[0196] In the present invention, the GDP-fucose synthase may be any
enzyme, so long as it is an enzyme relating to the synthesis of the
intracellular sugar nucleotide, GDP-fucose, as a supply source of
fucose to a sugar chain, and includes an enzyme which has influence
on the synthesis of the intracellular sugar nucleotide,
GDP-fucose.
[0197] The intracellular sugar nucleotide, GDP-fucose, is supplied
by a de novo synthesis pathway or a salvage synthesis pathway.
Thus, all enzymes relating to the synthesis pathways are included
in the GDP-fucose synthase.
[0198] The GDP-fucose synthase relating to the de novo synthesis
pathway includes GDP-mannose 4-dehydratase (hereinafter referred to
as "GMD"), GDP-keto-6-deoxymannose 3,5-epimerase, 4-reductase
(hereinafter referred to as "Fx") and the like.
[0199] The GDP-fucose synthase relating to the salvage synthesis
pathway includes GDP-beta-L-fucose pyrophosphorylase (hereinafter
referred to as "GFPP"), fucokinase and the like.
[0200] As the enzyme which has influence on the synthesis of an
intracellular sugar nucleotide, GDP-fucose, an enzyme which has
influence on the activity of the enzyme relating to the synthesis
pathway of the intracellular sugar nucleotide, GDP-fucose, and an
enzyme which has influence on the structure of substances as the
substrate of the enzyme are also included.
[0201] In the present invention, the GMD includes a protein encoded
by a DNA selected from the group consisting of (a) and (b), a
protein selected from the group consisting of (c), (d) and (e), and
the like:
[0202] (a) a DNA comprising the nucleotide sequence represented by
SEQ ID NO:65;
[0203] (b) a DNA which hybridizes with the DNA comprising the
nucleotide sequence represented by SEQ ID NO:65 under stringent
conditions and encodes a protein having GMD activity;
[0204] (c) a protein comprising the amino acid sequence represented
by SEQ ID NO:71;
[0205] (d) a protein which comprises an amino acid sequence in
which at least one amino acid is deleted, substituted, inserted
and/or added in the amino acid sequence represented by SEQ ID NO:71
and has GMD activity, and
[0206] (e) a protein which comprises an amino acid sequence having
a homology of 80% or more with the amino acid sequence represented
by SEQ ID NO:71 and has GMD activity.
[0207] Fx includes a protein encoded by a DNA selected from the
group consisting of (a) and (b), a protein selected from the group
consisting of (c), (d) and (e), and the like:
[0208] (a) a DNA comprising the nucleotide sequence represented by
SEQ ID NO:48;
[0209] (b) a DNA which hybridizes with the DNA comprising the
nucleotide sequence represented by SEQ ID NO:48 under stringent
conditions and encodes a protein having Fx activity;
[0210] (c) a protein comprising the amino acid sequence represented
by SEQ ID NO:19;
[0211] (d) a protein which comprises an amino acid sequence in
which at least one amino acid is deleted, substituted, inserted
and/or added in the amino acid sequence represented by SEQ ID NO:19
and has Fx activity, and
[0212] (e) a protein which comprises an amino acid sequence having
a homology of 80% or more with the amino acid sequence represented
by SEQ ID NO:19 and has Fx activity.
[0213] GFPP includes a protein encoded by a DNA selected from the
group consisting of (a) and (b), a protein selected from the group
consisting of (c), (d) and (e), and the like:
[0214] (a) a DNA comprising the nucleotide sequence represented by
SEQ ID NO:51;
[0215] (b) a DNA which hybridizes with the DNA comprising the
nucleotide sequence represented by SEQ ID NO:51 under stringent
conditions and encodes a protein having GFPP activity;
[0216] (c) a protein comprising the amino acid sequence represented
by SEQ ID NO:20;
[0217] (d) a protein which comprises an amino acid sequence in
which at least one amino acid is deleted, substituted, inserted
and/or added in the amino acid sequence represented by SEQ ID NO:20
and has GFPP activity, and
[0218] (e) a protein which comprises an amino acid sequence having
a homology of 80% or more with the amino acid sequence represented
by SEQ ID NO:20 and has GFPP activity.
[0219] In the present invention, the .alpha.1,6-fucose modifying
enzyme includes any enzyme, so long as it is an enzyme relating to
the reaction of binding of 1-position of fucose to 6-position of
N-acetylglucosamine in the reducing end through .alpha.-bond in the
complex N-glycoside-linked sugar chain. The enzyme relating to the
reaction of binding of 1-position of fucose to 6-position of
N-acetylglucosamine in the reducing end through .alpha.-bond in the
complex N-glycoside-linked sugar chain includes an enzyme which has
influence on the reaction of binding of 1-position of fucose to
6-position of N-acetylglucosamine in the reducing end through
.alpha.-bond in the complex N-glycoside-linked sugar chain.
[0220] The .alpha.1,6-fucose modifying enzyme includes
.alpha.1,6-fucosyltransferase, .alpha.-L-fucosidase and the
like.
[0221] Also, an enzyme which has influence on the activity of the
above enzyme relating to the reaction of binding of 1-position of
fucose to 6-position of N-acetylglucosamine in the reducing end
through .alpha.-bond in the complex N-glycoside-linked sugar chain
and an enzyme which has influence on the structure of substances as
the substrate of the enzyme are included.
[0222] The .alpha.1,6-fucosyltransferase includes a protein encoded
by a DNA selected from the group consisting of the following (a),
(b), (c) and (d), a protein selected from the group consisting of
the following (e), (f), (g), (h), (i) and (j), and the like:
[0223] (a) a DNA comprising the nucleotide sequence represented by
SEQ ID NO:1;
[0224] (b) a DNA comprising the nucleotide sequence represented by
SEQ ID NO:2;
[0225] (c) a DNA which hybridizes with the DNA comprising the
nucleotide sequence represented by SEQ ID NO:1 under stringent
conditions and encodes a protein having
.alpha.1,6-fucosyltransferase activity;
[0226] (d) a DNA which hybridizes with the DNA comprising the
nucleotide sequence represented by SEQ ID NO:2 under stringent
conditions and encodes a protein having
.alpha.1,6-fucosyltransferase activity;
[0227] (e) a protein comprising the amino acid sequence represented
by SEQ ID NO:23;
[0228] (f) a protein comprising the amino acid sequence represented
by SEQ ID NO:24;
[0229] (g) a protein which comprises an amino acid sequence in
which at least one amino acid is deleted, substituted, inserted
and/or added in the amino acid sequence represented by SEQ ID NO:23
and has .alpha.1,6-fucosyltransferase activity;
[0230] (h) a protein which comprises an amino acid sequence in
which at least one amino acid is deleted, substituted, inserted
and/or added in the amino acid sequence represented by SEQ ID NO:24
and has .alpha.1,6-fucosyltransferase activity;
[0231] (i) a protein which comprises an amino acid sequence having
a homology of 80% or more with the amino acid sequence represented
by SEQ ED NO:23 and has .alpha.1,6-fucosyltransferase activity,
and
[0232] (j) a protein which comprises an amino acid sequence having
a homology of 80% or more with the amino acid sequence represented
by SEQ ID NO:24 and has .alpha.1,6-fucosyltransferase activity.
[0233] The GDP-fucose transport protein may be any protein, so long
as it is a protein relating to the transportation of the
intracellular sugar nucleotide, GDP-fucose to the Golgi body or a
protein which has an influence on the reaction to transport the
intracellular sugar nucleotide, GDP-fucose to the Golgi body.
[0234] The GDP-fucose transport protein includes a GDP-fucose
transporter and the like.
[0235] Furthermore, the protein which has an influence on the
reaction to transport the intracellular sugar nucleotide,
GDP-fucose to the Golgi body includes a protein which has an
influence on the above GDP-fucose transport protein or has an
influence on the expression thereof.
[0236] In the present invention, the GDP-fucose transporter
includes a protein encoded by a DNA selected from the group
consisting of the following (a) to (h), and the like:
[0237] (a) a DNA comprising the nucleotide sequence represented by
SEQ ID NO:91;
[0238] (b) a DNA comprising the nucleotide sequence represented by
SEQ ID NO:93;
[0239] (c) a DNA comprising the nucleotide sequence represented by
SEQ ID NO:95;
[0240] (d) a DNA comprising the nucleotide sequence represented by
SEQ ID NO:97;
[0241] (e) a DNA which hybridizes with the DNA comprising the
nucleotide sequence represented by SEQ ID NO:91 under stringent
conditions and encodes a protein having GDP-fucose transporter
activity;
[0242] (f) a DNA which hybridizes with the DNA comprising the
nucleotide sequence represented by SEQ ID NO:93 under stringent
conditions and encodes a protein having GDP-fucose transporter
activity;
[0243] (g) a DNA which hybridizes with the DNA comprising the
nucleotide sequence represented by SEQ ID NO:95 under stringent
conditions and encodes a protein having GDP-fucose transporter
activity; and
[0244] (h) a DNA which hybridizes with the DNA comprising the
nucleotide sequence represented by SEQ ID NO:97 under stringent
conditions and encodes a protein having GDP-fucose transporter
activity.
[0245] Furthermore, the GDP-fucose transporter of the present
invention includes a protein selected from the group consisting of
the following (i) to (t), and the like:
[0246] (i) a protein comprising the amino acid sequence represented
by SEQ ID NO:92;
[0247] (j) a protein comprising the amino acid sequence represented
by SEQ ID NO:94;
[0248] (k) a protein comprising the amino acid sequence represented
by SEQ ID NO:96;
[0249] (l) a protein comprising the amino acid sequence represented
by SEQ ID NO:98;
[0250] (m) a protein which comprises an amino acid sequence in
which at least one amino acid is deleted, substituted, inserted
and/or added in the amino acid sequence represented by SEQ ID NO:92
and has GDP-fucose transporter activity,
[0251] (n) a protein which comprises an amino acid sequence in
which at least one amino acid is deleted, substituted, inserted
and/or added in the amino acid sequence represented by SEQ ID NO:94
and has GDP-fucose transporter activity,
[0252] (o) a protein which comprises an amino acid sequence in
which at least one amino acid is deleted, substituted, inserted
and/or added in the amino acid sequence represented by SEQ ID NO:96
and has GDP-fucose transporter activity,
[0253] (p) a protein which comprises an amino acid sequence in
which at least one amino acid is deleted, substituted, inserted
and/or added in the amino acid sequence represented by SEQ ID NO:98
and has GDP-fucose transporter activity,
[0254] (q) a protein which comprises an amino acid sequence having
a homology of 80% or more with the amino acid sequence represented
by SEQ ID NO:92 and has GDP-fucose transporter activity,
[0255] (r) a protein which comprises an amino acid sequence having
a homology of 80% or more with the amino acid sequence represented
by SEQ ID NO:94 and has GDP-fucose transporter activity,
[0256] (s) a protein which comprises an amino acid sequence having
a homology of 80% or more with the amino acid sequence represented
by SEQ ID NO:96 and has GDP-fucose transporter activity, and
[0257] (t) a protein which comprises an amino acid sequence having
a homology of 80% or more with the amino acid sequence represented
by SEQ ID NO:98 and has GDP-fucose transporter activity.
[0258] A DNA which hybridizes under stringent conditions is a DNA
obtained, e.g., by a method such as colony hybridization, plaque
hybridization or Southern blot hybridization using a DNA such as
the DNA having the nucleotide sequence represented by SEQ ID NO:1,
2, 48, 51, 65, 91, 93, 95 or 97 or a partial fragment thereof as
the probe, and specifically includes a DNA which can be identified
by carrying out hybridization at 65.degree. C. in the presence of
0.7 to 1.0 mol/l sodium chloride using a filter to which colony- or
plaque-derived DNAs are immobilized, and then washing the filter at
65.degree. C. using 0.11 to 2.times.SSC solution (composition of
the 1.times.SSC solution comprising 150 mmol/l sodium chloride and
15 mmol/l sodium citrate). The hybridization can be carried out in
accordance with the methods described, e.g., in Molecular Cloning,
A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press
(1989) (hereinafter referred to as "Molecular Cloning, Second
Edition"), Current Protocols in Molecular Biology, John Wiley &
Sons, 1987-1997 (hereinafter referred to as "Current Protocols in
Molecular Biology"); DNA Cloning 1: Core Techniques, A Practical
Approach, Second Edition, Oxford University (1995) and the like.
The hybridizable DNA includes a DNA having at least 60% or more,
preferably 70% or more, more preferably 80% or more, still more
preferably 90% or more, far more preferably 95% or more, and most
preferably 98% or more, of homology with the nucleotide sequence
represented by SEQ ID NO:1, 2, 48, 51, 65, 91, 93, 95 or 97.
[0259] The protein which comprises an amino acid sequence in which
at least one amino acid is deleted, substituted, inserted and/or
added in the amino acid sequence represented by SEQ ID NO:19, 20,
23, 24, 71, 92, 94, 96 or 98 and has .alpha.1,6-fucosyltransferase
activity, GMD activity, Fx activity, GFPP activity or GFP-fucose
transporter activity can be obtained, e.g., by introducing a
site-directed mutation into a DNA encoding a protein having the
amino acid sequence represented by SEQ ID NO:1, 2, 48, 51 or 65,
respectively, using the site-directed mutagenesis described, e.g.,
in Molecular Cloning, Second Edition; Current Protocols in
Molecular Biology; Nucleic Acids Research, 10, 6487 (1982); Proc.
Natl. Acad. Sci. USA, 79, 6409 (1982); Gene, 34, 315 (1985);
Nucleic Acids Research, 13, 4431 (1985); Proc. Natl. Acad. Sci.
USA, 82, 488 (1985); and the like. The number of amino acids to be
deleted, substituted, inserted and/or added and the number is not
particularly limited, but is a number which can be deleted,
substituted or added by a known technique such as the site-directed
mutagenesis, e.g., it is 1 to several tens, preferably 1 to 20,
more preferably 1 to 10, and most preferably 1 to 5.
[0260] Also, in order to maintain the .alpha.1,6-fucosyltransferase
activity, GMD activity, Fx activity, GFPP activity or GDP-fucose
transporter activity of the protein to be used in the present
invention, it has at least 80% or more, preferably 85% or more,
more preferably 90% or more, still more preferably 95% or more, far
more preferably 97% or more, and most preferably 99% or more, of
homology with the amino acid sequence represented by SEQ ID NO:19,
20, 23, 24, 71, 92, 94, 96 or 98, when calculated using an
analyzing soft such as BLAST [J. Mol. Biol., 215, 403 (1990)] of
FASTA [Methods in Enzymology, 183, 63 (1990)].
[0261] As the method for obtaining the above cells, any technique
can be used, so long as it can decrease or delete the enzyme
activity of interest. The technique for decreasing or deleting the
enzyme activity includes:
[0262] (a) a gene disruption technique which comprises targeting a
gene encoding the enzyme;
[0263] (b) a technique for introducing a dominant negative mutant
of a gene encoding the enzyme;
[0264] (c) a technique for introducing mutation into the enzyme,
and
[0265] (d) a technique for surprising transcription and/or
translation of a gene encoding the enzyme, and the like.
[0266] Also, the method includes a method for selecting a cell
having resistance to lectin which recognizes the structure of a
sugar chain in which 1-position of fucose is bound to 6-position of
N-acetylglucosamine in the reducing end through .alpha.-bond in a
complex N-glycoside-linked sugar chain.
[0267] The growth of lectin-resistant cell is not inhibited in the
presence of a lectin at an effective concentration during cell
culturing.
[0268] In the present invention, the effective concentration of a
lectin which does not inhibit the growth can be decided depending
on the cell line, and is generally 10 .mu.g/ml to 10.0 mg/ml,
preferably 0.5 to 2.0 mg/ml. The effective concentration of lectin
in the case where mutation is introduced into a parent cell is a
concentration in which the parent cell cannot normally grow or
higher than the concentration, and is a concentration which is
preferably similar to, more preferably 2 to 5 times, still more
preferably 10 times, and most preferably 20 times or more, higher
than the concentration in which the parent cell cannot normally
grow.
[0269] The lectin which recognizes a sugar chain structure in which
1-position of fucose is bound to 6-position of N-acetylglucosamine
through .alpha.-bond includes any lectin, so long as it is a lectin
which is capable of recognizing the sugar chain structure. Examples
include lentil agglutinin derived from Lens culinaris (Lens
culinaris lectin LCA), pea lectin derived from Pisum sativuma (pea
lectin PSA), (agglutinin derived from Vicia faba (broad bean lectin
VFA), lectin derived from Aleuria aurantia (Aleuria aurantia lectin
AAL) and the like.
[0270] The parent cell is a cell before a certain treatment is
applied, namely a cell before the step for selecting the
lectin-resistant cell used in the present invention is carried out
or a cell before genetic engineering techniques for decreasing or
deleting the above enzyme activity is carried out.
[0271] Although the parent cell is not particularly limited, the
following cells are exemplified.
[0272] The parent cell of NS0 cell includes NS0 cells described in
literatures such as BIO/TECHNOLOGY, 10, 169 (1992) and Biotechnol.
Bioeng., 73, 261 (2001). Furthermore, it includes NS0 cell line
(RCB 0213) registered at RIKEN Cell Bank, The Institute of Physical
and Chemical Research, sub-cell lines obtained by acclimating these
cell lines to media in which they can grow, and the like.
[0273] The parent cell of SP2/0-Ag14 cell includes SP2/0-Ag14 cells
described in literatures such as J. Immunol., 126, 317 (1981),
Nature, 276, 269 (1978) and Human Antibodies and Hybridomas, 3, 129
(1992). Furthermore, it includes SP2/0-Ag14 cell (ATCC CRL-1581)
registered at ATCC, sub-cell lines obtained by naturalizing these
cell lines to media in which they can grow (ATCC CRL-1581.1), and
the like.
[0274] The parent cell of CHO cell derived from Chinese hamster
ovary tissue includes CHO cells described in literatures such as
Journal of Experimental Medicine, 108, 945 (1958), Proc. Natl.
Acad. Sci. USA, 60, 1275 (1968), Genetics, 55, 513 (1968),
Chromosoma, 41, 129 (1973), Methods in Cell Science, 18, 115
(1996), Radiation Research, 148, 260 (1997), Proc. Natl. Acad. Sci.
USA, 77, 4216 (1980), Proc. Natl. Acad. Sci. USA, 6, 1275 (1968),
Cell, 6, 121 (1975) and Molecular Cell Genetics, Appendix I, II (p.
883-900). Furthermore, it includes cell line CHO-K1 (ATCC CCL-61),
cell line DUXB11 (ATCC CRL-9060) and cell line Pro-5 (ATCC
CRL-1781) registered at ATCC, commercially available cell line
CHO-S (Cat # 11619 of Life Technologies), sub-cell lines obtained
by acclimating these cell lines to media in which they can grow,
and the like.
[0275] The parent cell of a rat myeloma cell line
YB2/3HL.P2.G11.16Ag.20 cell includes cell lines established from
Y3/Ag1.2.3 cell (ATCC CRL-1631) such as YB2/3HL.P2.G11.16Ag.20 cell
described in literatures such as J. Cell. Biol., 93, 576 (1982) and
Methods Enzymol. 73B, 1 (1981). Furthermore, it include
YB2/3HL.P2.G11.16Ag.20 cell (ATCC CRL-1662) registered at ATCC,
sub-lines obtained by acclimating these cell lines to media in
which they can grow, and the like.
[0276] In the present invention, Fc.gamma.R means an Fc receptor
(hereinafter also referred to as "FcR") against an IgG class
antibody. FcR means a receptor which binds to the Fc region of an
antibody [Annu. Rev. Immunol., 2, 457 (1991)]. Furthermore,
Fc.gamma.R includes Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII
subclasses and their allele mutants and isoforms formed by
alternative splicing. In addition, Fc.gamma.RII includes
Fc.gamma.RIIa and Fc.gamma.RIIb, and Fc.gamma.RII includes
Fc.gamma.RIIIa and Fc.gamma.RIIIb [Annu. Rev. Immunol., 9, 457
(1991)].
[0277] The binding activity to Fc.gamma.RIIIa can be increased by
binding sugar chains to the Fc region of an antibody molecule so as
to adjust the ratio of a sugar chain in which fucose is not bound
to N-acetylglucosamine in the reducing end in the sugar chain among
the total complex N-glycoside-linked sugar chains bound to the Fc
region to preferably 20% or more, more preferably 30% or more,
still more preferably 40% or more, particularly preferably 50% or
more, and most preferably 100%.
[0278] The ratio of a sugar chain in which fucose is not bound to
N-acetylglucosamine in the reducing end in the sugar chain among
the total complex N-glycoside-linked sugar chains bound to the Fc
region contained in the antibody composition is a ratio of the
number of a sugar chain in which fucose is not bound to
N-acetylglucosamine in the reducing end in the sugar chain to the
total number of the complex N-glycoside-linked sugar chains bound
to the Fc region contained in the composition. Also, the ratio of a
sugar chain is preferably a ratio of a sugar chain in which
1-position of fucose is not bound to 6-position of
N-acetylglucosamine in the reducing end through .alpha.-bond in the
sugar chain.
[0279] The sugar chain in which fucose is not bound to
N-acetylglucosamine in the reducing end in the complex
N-glycoside-linked sugar chain is a sugar chain in which fucose is
not bound to AF-acetylglucosamine in the reducing end through
.alpha.-bond in the complex N-glycoside-linked sugar chain.
Preferably, it is a sugar chain in which 1-position of fucose is
not bound to 6-position of N-acetylglucosamine in the complex
N-glycoside-linked sugar chain through .alpha.-bond.
[0280] The ratio of a sugar chain in which fucose is not bound to
N-acetylglucosamine in the reducing end in the sugar chain
contained in the composition which comprises an antibody molecule
having complex N-glycoside-linked sugar chains in the Fc region can
be determined by releasing the sugar chain from the antibody
molecule using a known method such as hydrazinolysis, enzyme
digestion or the like [Biochemical Experimentation Methods
23--Method for Studying Glycoprotein Sugar Chain (Japan Scientific
Societies Press), edited by Reiko Takahashi (1989)), carrying out
fluorescence labeling or radioisotope labeling of the released
sugar chain, and then separating the labeled sugar chain by
chromatography. Also, the released sugar chain can be determined by
analyzing it with the HPAED-PAD method [J. Liq. Chromatogr., 6,
1577 (1983)]. The antibody composition in which binding activity to
Fc.gamma.RIIa has been enhanced by the method of the present
invention has high ADCC activity.
[0281] In the present invention, the ADCC activity is a cytotoxic
activity in which an antibody bound to a cell surface antigen on a
cell such as a tumor cell in the living body activates an effector
cell mediated the antibody Fc region and an Fc receptor existing on
effector cell surface and thereby injures the tumor cell and the
like [Monoclonal Antibodies: Principles and Applications,
Wiley-Liss, Inc., Chapter 2.1 (1995)]. The effector cell includes
killer cells, natural killer cells, monocytes, macrophages, and the
like.
[0282] A process for producing a host cell in which the activity of
a protein relating to modification of a sugar chain in which fucose
is bound to N-acetylglucosamine in the reducing end in the complex
NV-glycoside-linked sugar chain is decreased or deleted used in the
method of the present invention is explained below in detail.
[0283] 1. Preparation of Host Cell Used in the Method of the
Invention
[0284] The host cell used in the method of the present invention
can be prepared by the following techniques.
[0285] (1) Gene Disruption Technique which Comprises Targeting a
Gene Encoding an Enzyme
[0286] The host cell used in the method of the present invention
can be prepared by targeting a gene encoding a GDP-fucose synthase,
.alpha.1,6-fucose modifying enzyme or a GDP-fucose transport
protein by using a gene disruption technique. The GDP-fucose
synthase includes GMD, Fx, GFPP, fucokinase and the like. The
.alpha.1,6-fucose modifying enzyme includes
.alpha.1,6-fucosyltransferase, .alpha.-L-fucosidase and the like.
The GDP-fucose transport protein includes GDP-fucose
transporter.
[0287] The gene as used herein includes DNA and RNA.
[0288] The gene disruption method may be any method, so long as it
can disrupt the gene encoding the target enzyme. Examples include
an antisense method, a ribozyme method, a homologous recombination
method, an RNA-DNA oligonucleotide (RDO) method, an RNA
interference (RNAi) method, a method using retrovirus, a method
using transposon and the like. The methods are specifically
described below.
[0289] (a) Preparation of Host Cell Used in the Present Invention
by the Antisense Method or the Ribozyme Method
[0290] The host cell used in the method of the present invention
can be prepared by targeting the GDP-fucose synthase,
.alpha.1,6-fucose modifying enzyme or the GDP-fucose transport
protein according to the antisense or ribozyme method described in
Cell Technology, 12, 239 (1993); BIO/TECHNOLOGY, 17, 1097 (1999),
Hum. Mol Genet., 5, 1083 (1995); Cell Technology, 13, 255 (1994);
Proc. Natl. Acad. Sci. USA, 96, 1886 (1999); or the like, e.g., in
the following manner.
[0291] A cDNA or a genomic DNA encoding GDP-fucose synthase,
.alpha.1,6-fucose modifying enzyme or the GDP-fucose transport
protein is prepared.
[0292] The nucleotide sequence of the prepared cDNA or genomic DNA
is determined.
[0293] Based on the determined DNA sequence, an antisense gene or
ribozyme construct of an appropriate length comprising a DNA moiety
which encodes the GDP-fucose synthase, .alpha.1,6-fucose modifying
enzyme or the GDP-fucose transport protein, a part of its
untranslated region or an intron is designed.
[0294] In order to express the antisense gene or ribozyme in a
cell, a recombinant vector is prepared by inserting a fragment or
total length of the prepared DNA into downstream of the promoter of
an appropriate expression vector.
[0295] A transformant is obtained by introducing the recombinant
vector into a host cell suitable for the expression vector.
[0296] The host cell used in the method of the present invention
can be obtained by selecting a transformant based on the activity
of the GDP-fucose synthase, the .alpha.1,6-fucose modifying enzyme
or the GDP-fucose transport protein. The host cell of the present
invention can also be obtained by selecting a transformant based on
the sugar chain structure of a glycoprotein on the cell membrane or
the sugar chain structure of the produced antibody molecule.
[0297] As the host cell used for preparing the host cell used in
the method of the present invention, any cell such as yeast, an
animal cell, an insect cell or a plant cell can be used, so long as
it has a gene encoding the target GDP-fucose synthase,
.alpha.1,6-fucose modifying enzyme or GDP-fucose transport protein.
Examples include host cells described in the following item 3.
[0298] As the expression vector, a vector which is autonomously
replicable in the host cell or can be integrated into the
chromosome and comprises a promoter at such a position that the
designed antisense gene or ribozyme can be transferred is used.
Examples include expression vectors described in the following item
3.
[0299] As the method for introducing a gene into various host
cells, the methods for introducing recombinant vectors suitable for
various host cells described in the following item 3, can be
used.
[0300] The method for selecting a transform ant based on the
activity of the GDP-fucose synthase, the .alpha.1,6-fucose
modifying enzyme or the GDP-fucose transport protein includes
biochemical methods or genetic engineering techniques described in
New Biochemical Experimentation Series (Shin-Jikken Kagakii Koza)
3--Saccharides (Toshitsu) I, Glycoprotein (Totanpakushitu) (Tokyo
Kagaku Dojin), edited by Japanese Biochemical Society (1988); Cell
Engineering (Saibo Kogaku). Supplement, Experimental Protocol
Series, Glycobiology Experimental Protocol, Glycoprotein,
Glycolipid and Proteoglycan (Shujun-sha), edited by Naoyuki
Taniguchi, Akemi Suzuki, Kiyoshi Furukawa and Kazuyuki Sugawara
(1996); Molecular Cloning, Second Edition; Current Protocols in
Molecular Biology; and the like. The biochemical method includes a
method in which the enzyme activity is evaluated using
enzyme-specific substrate and the like. The genetic engineering
technique include the Northern analysis, RT-PCR and the like which
measures the amount of mRNA of a gene encoding the enzyme.
[0301] The method for selecting a transformant based on the sugar
chain structure of a glycoprotein on the cell membrane includes the
methods described in the following item 1(5). The method for
selecting a transformant based on the sugar chain structure of a
produced antibody molecule includes the methods described in the
following items 6 and 7.
[0302] As the method for preparing cDNA encoding the GDP-fucose
synthase, .alpha.1,6-fucose modifying enzyme or the GDP-fucose
transport protein, the following method is exemplified.
[0303] Preparation of DNA:
[0304] A total RNA or RNA is prepared from a human or non-human
animal tissue or cell.
[0305] The mRNA of a human or non-human tissue or cell may be a
commercially available product (e.g., manufactured by Clontech) or
may be prepared from a human or non-human animal tissue or cell as
follows. The method for preparing a total RNA from a human or
non-human animal tissue or cell includes the guanidine
thiocyanate-cesium trifluoroacetate method [Methods in Enzymology,
154, 3 (1987)], the acidic guanidine thiocyanate phenol chloroform
(AGPC) method [Analytical Biochemistry, 162, 156 (1987);
Experimental Medicine, 9, 1937 (1991)] and the like.
[0306] Also, the mRNA can be prepared as poly(A).sup.+ RNA from a
total RNA by the oligo(dT)-immobilized cellulose column method
(Molecular Cloning, Second Edition) and the like.
[0307] In addition, mRNA can be prepared using a kit such as Fast
Track mRNA Isolation Kit (manufactured by Invitrogen) or Quick Prep
mRNA Purification Kit (manufactured by Pharmacia).
[0308] A cDNA library is prepared from the prepared mRNA of a human
or non-human animal tissue or cell. The method for preparing cDNA
library includes the methods described in Molecular Cloning, Second
Edition; Current Protocols in Molecular Biology; and the like, or
methods using a commercially available kit such as SuperScript
Plasmid System for cDNA Synthesis and Plasmid Cloning (manufactured
by Life Technologies) or ZAP-cDNA Synthesis Kit (manufactured by
STRATAGENE).
[0309] As the cloning vector for the preparation of the cDNA
library, any vector such as a phage vector or a plasmid vector can
be used, so long as it is autonomously replicable in Escherichia
coli K12. Examples include ZAP Express [manufactured by STRATAGENE,
Strategies, 5, 58 (1992)], pBluescript II SK(+) [Nucleic Acids
Research, 172, 9494 (1989)], Lambda ZAP II (manufactured by
STRATAGENE), .lambda.gt10 ad .lambda.gt11 [DNA Cloning, A Practical
Approach, 1, 49 (1985)], .lambda.TriplEx (manufactured by
Clontech), .lambda.ExCell (manufactured by Pharmacia), pT7T318U
(manufactured by Pharmacia), pcD2 [Mol. Cell. Biol., 3, 280
(1983)], pUC18 [Gene 33, 103 (1985)) and the like.
[0310] Any microorganism can be used as the host microorganism for
the preparation of the DNA library, and Escherichia coli is
preferably used. Examples include Escherichia coli XL1-Blue MRF'
[manufactured by STRATAGENE, Strategies, 5, 81 (1992)], Escherichia
coli C600 [Genetics, 39440 (1954)], Escherichia coli Y1088
[Science, 222, 778 (1983)], Escherichia coli Y1090 [Science, 222,
778 (1983)], Escherichia coli NM522 [J. Mol. Biol., 166, 1 (1983)],
Escherichia coli K802 [J. Mol. Biol., 16, 118 (1966)], Escherichia
coli JM105 [Gene, 38, 275 (1985)] and the like.
[0311] The cDNA library can be used as such in the following
analysis, and in order to obtain a full length cDNA as efficient as
possible by decreasing the ratio of an infull length cDNA, a cDNA
library prepared by using the oligo cap method developed by Sugano
et al. [Gene, 138, 171 (1994), Gene, 200, 149 (1997)] Protein,
Nucleic Acid and Protein 41, 603 (1996)], Experimental Medicine,
12491 (1993); cDNA Cloning (Yodo-sha) (1996), Methods for Preparing
Gene Libraries (Yodo-sha) (1994)] can be used in the following
analysis.
[0312] Based on the amino acid sequence of the GDP-fucose synthase,
the .alpha.1,6-fucose modifying enzyme or the GDP-fucose transport
protein, degenerative primers specific for the 4'-terminal and
3'-terminal nucleotide sequences of a nucleotide sequence presumed
to encode the amino acid sequence are prepared, and DNA is
amplified by PCR [PCR Protocols, Academic Press (1990)] using the
prepared cDNA library as the template to obtain a gene fragment
encoding the GDP-fucose synthase, the .alpha.1,6-fucose modifying
enzyme or the GDP-fucose transport protein.
[0313] It can be confirmed that the obtained gene fragment is a DNA
encoding the GDP-fucose synthase, the .alpha.1,6-fucose modifying
enzyme or the GDP-fucose transport protein by a method generally
used for analyzing a nucleotide such as the dideoxy method of
Sanger et al. [Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)] or by
using a nucleotide sequence analyzer such as ABIPRISM 377 DNA
Sequencer (manufactured by PE Biosystems).
[0314] A DNA encoding the GDP-fucose synthase, the
.alpha.1,6-fucose modifying enzyme or the GDP-fucose transport
protein can be obtained by carrying out colony hybridization or
plaque hybridization (Molecular Cloning, Second Edition) for the
cDNA or cDNA library synthesized from the mRNA contained in the
human or non-human animal tissue or cell, using the gene fragment
as a DNA probe.
[0315] Also, using the primers used for obtaining the gene fragment
encoding the GDP-fucose synthase, the .alpha.1,6-fucose modifying
enzyme or the GDP-fucose transport protein, a DNA encoding the
GDP-fucose synthase, the .alpha.1,6-fucose modifying enzyme or the
GDP-fucose transport protein can also be obtained by carrying out
screening by PCR using the cDNA or cDNA library synthesized from
the rRNA contained in a human or non-human animal tissue or cell as
the template.
[0316] The nucleotide sequence of the obtained DNA encoding the
GDP-fucose synthase, the .alpha.1,6-fucose modifying enzyme or the
GDP-fucose transport protein is analyzed from its terminus and
determined by a method generally used for analyzing a nucleotide
such as the dideoxy method of Sanger et al. [Proc. Natl. Acad. Sci.
USA, 74, 5463 (1977)] or by using a nucleotide sequence analyzer
such as ABIPRISM 377 DNA Sequencer (manufactured by PE
Biosystems).
[0317] A gene encoding the GDP-fucose synthase, the
.alpha.1,6-fucose modifying enzyme or the GDP-fucose transport
protein can also be determined from genes in data bases by
searching nucleotide sequence data bases such as GenBank, EMBL and
DDBJ using a homology retrieving program such as BLAST based on the
determined cDNA nucleotide sequence.
[0318] The nucleotide sequence of the gene encoding the GDP-fucose
synthase obtained by the above method includes the nucleotide
sequence represented by SEQ ID NO:48, 51 or 65. The nucleotide
sequence of the gene encoding the .alpha.1,6-fucose modifying
enzyme includes the nucleotide sequence represented by SEQ ID NO:1
or 2. The nucleotide sequence of the gene encoding the GDP-fucose
transport protein includes the nucleotide sequence represented by
SEQ ID NO:91, 93, 95 or 97.
[0319] The cDNA encoding the GDP-fucose synthase, the
.alpha.1,6-fucose modifying enzyme or the GDP-fucose transport
protein can also be obtained by chemically synthesizing it with a
DNA synthesizer such as DNA Synthesizer model 392 manufactured by
Perkin Elmer using the phosphoamidite method, based on the
determined DNA nucleotide sequence.
[0320] The method for preparing a genomic DNA encoding the
GDP-fucose synthase, the .alpha.1,6-fucose modifying enzyme or the
GDP-fucose transport protein includes known methods described in
Molecular Cloning, Second Edition; Current Protocols in Molecular
Biology; and the like. Furthermore, the genomic DNA can be prepared
by using a kit such as Genome DNA Library Screening System
(manufactured by Genome Systems) or Universal GenomeWalker.TM. Kits
(manufactured by CLONTECH).
[0321] The nucleotide sequence of the genomic DNA encoding the
GDP-fucose synthase obtained by the method includes the nucleotide
sequence represented by SEQ ID NO:67 or 70. The nucleotide sequence
of the genomic DNA encoding the .alpha.1,6-fucose modifying enzyme
includes the nucleotide sequence represented by SEQ ID NO:3. The
nucleotide sequence of the genomic DNA encoding the GDP-fucose
transport protein includes the nucleotide sequence represented by
SEQ ID NO:99 or 100.
[0322] In addition, the host cell can also be obtained without
using an expression vector, by directly introducing an antisense
oligonucleotide or ribozyme into a host cell, which is designed
based on the nucleotide sequence encoding the GDP-fucose synthase,
the .alpha.1,6-fucose modifying enzyme or the GDP-fucose transport
protein.
[0323] The antisense oligonucleotide or ribozyme can be prepared in
the usual method or by using a DNA synthesizer. Specifically, it
can be prepared based on the sequence information of an
oligonucleotide having a corresponding sequence of continued 5 to
150 bases, preferably 5 to 60 bases, and more preferably 5 to 40
bases, among nucleotide sequences of a cDNA and a genomic DNA
encoding the GDP-fucose synthase, the .alpha.1,6-fucose modifying
enzyme or the GDP-fucose transport protein by synthesizing an
oligonucleotide which corresponds to a sequence complementary to
the oligonucleotide (antisense oligonucleotide) or a ribozyme
comprising the oligonucleotide sequence.
[0324] The oligonucleotide includes oligo RNA and derivatives of
the oligonucleotide (hereinafter referred to as "oligonucleotide
derivatives").
[0325] The oligonucleotide derivatives includes oligonucleotide
derivatives in which a phosphodiester bond in the oligonucleotide
is converted into a phosphorothioate bond, an oligonucleotide
derivative in which a phosphodiester bond in the oligonucleotide is
converted into an N3'-P5' phosphoamidate bond, an oligonucleotide
derivative in which ribose and a phosphodiester bond in the
oligonucleotide are converted into a peptide-nucleic acid bond, an
oligonucleotide derivative in which uracil in the oligonucleotide
is substituted with C-5 propynyluracil, an oligonucleotide
derivative in which uracil in the oligonucleotide is substituted
with C-5 thiazoleuracil, an oligonucleotide derivative in which
cytosine in the oligonucleotide is substituted with C-5
propynylcytosine, an oligonucleotide derivative in which cytosine
in the oligonucleotide is substituted with phenoxazine-modified
cytosine, an oligonucleotide derivative in which ribose in the
oligonucleotide is substituted with 2'-O-propylribose and an
oligonucleotide derivative in which ribose in the oligonucleotide
is substituted with 2'-methoxyethoxyribose [Cell Technology (Saibo
Kogaku), 16, 1463 (1997)].
[0326] (b) Preparation of Host Cell Used in the Method of the
Present Invention by Homologous Recombination
[0327] The host cell used in the method of the present invention
can be prepared by targeting a gene encoding the GDP-fucose
synthase, the .alpha.1,6-fucose modifying enzyme or the GDP-fucose
transport protein and modifying the target gene on chromosome
through a homologous recombination technique.
[0328] The target gene on the chromosome can be modified by using a
method described in Manipulating the Mouse Embryo, A Laboratory
Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994)
(hereinafter referred to as "Manipulating the Mouse Embryo, A
Laboratory Manual"), Gene Targeting, A Practical Approach, IRL
Press at Oxford University Press (1993); Biomanual Series 8, Gene
Targeting, Preparation of Mutant Mice using ES, Yodo-sha (1995)
(hereinafter referred to as "Preparation of Mutant Mice using ES
Cells"); or the like, for example, as follows.
[0329] A genomic DNA encoding the GDP-fucose synthase, the
.alpha.1,6-fucose modifying enzyme or the GDP-fucose transport
protein is prepared.
[0330] Based on the nucleotide sequence of the genomic DNA, a
target vector is prepared for homologous recombination of a target
gene to be modified (e.g., structural gene encoding the GDP-fucose
synthase, the .alpha.1,6-fucose modifying enzyme or the GDP-fucose
transport protein or a promoter gene).
[0331] The host cell used in the method of the present invention
can be produced by introducing the prepared target vector into a
host cell and selecting a cell in which homologous recombination
occurred between the target gene and target vector.
[0332] As the host cell, any cell such as yeast, an animal cell, an
insect cell or a plant cell can be used, so long as it has a gene
encoding the GDP-fucose synthase, the .alpha.1,6-fucose modifying
enzyme or the GDP-fucose transport protein. Examples include the
host cells described in the following item 3.
[0333] The method for preparing a genomic DNA encoding the
GDP-fucose synthase, the .alpha.1,6-fucose modifying enzyme or the
GDP-fucose transport protein includes the methods described in
"Preparation method of genomic DNA" in the item 1(1)(a) and the
like.
[0334] The nucleotide sequence of the genomic DNA encoding the
GDP-fucose synthase obtained by the above method includes the
nucleotide sequence represented by SEQ ID NO:67 or 70. The
nucleotide sequence of the genomic DNA of the .alpha.1,6-fucose
modifying enzyme includes the nucleotide sequence represented by
SEQ ID NO:3. The nucleotide sequence of the genomic DNA of the
GDP-fucose transport protein includes the nucleotide sequence
represented by SEQ ID NO:99 or 100.
[0335] The target vector for the homologous recombination of the
target gene can be prepared in accordance with a method described
in Gene Targeting, A Practical Approach, IRL Press at Oxford
University Press (1993); Preparation of Mutant Mice using ES Cells;
or the like. The target vector can be used as either a replacement
type or an insertion type.
[0336] For introducing the target vector into various host cells,
the methods for introducing recombinant vectors suitable for
various host cells described in the following item 3 can be
used.
[0337] The method for efficiently selecting a homologous
recombinant includes a method such as the positive selection,
promoter selection, negative selection or polyA selection described
in Gene Targeting, A Practical Approach, IRL Press at Oxford
University Press (1993); Preparation of Mutant Mice using ES Cells;
or the like. The method for selecting the homologous recombinant of
interest from the selected cell lines includes the Southern
hybridization method for genomic DNA (Molecular Cloning, Second
Edition), PCR [PCR Protocols, Academic Press (1990)], and the
like.
[0338] (c) Preparation of Cell of the Present Invention by RDO
Method
[0339] The host cell used in the method of the present invention
can be prepared by targeting a gene encoding the GDP-fucose
synthase, the .alpha.1,6-fucose modifying enzyme or the GDP-fucose
transport protein according to an RDO method, for example, as
follows.
[0340] A cDNA or a genomic DNA encoding the GDP-fucose synthase,
the .alpha.1,6-fucose modifying enzyme or the GDP-fucose transport
protein is prepared.
[0341] The nucleotide sequence of the prepared cDNA or genomic DNA
is determined.
[0342] Based on the determined DNA sequence, an RDO construct of an
appropriate length comprising a DNA encoding the GDP-fucose
synthase, the .alpha.1,6-fucose modifying enzyme or the GDP-fucose
transport protein, a DNA encoding a untranslated region or a DNA
encoding an intron, is designed and synthesized.
[0343] The host cell used in the method of the present invention
can be obtained by introducing the synthesized RDO into a host cell
and then selecting a transformant in which a mutation occurred in
the target enzyme, i.e., the GDP-fucose synthase, the
.alpha.1,6-fucose modifying enzyme or the GDP-fucose transport
protein.
[0344] As the host cell, any cell such as yeast, an animal cell, an
insect cell or a plant cell can be used, so long as it has a gene
encoding the target GDP-fucose synthase, .alpha.1,6-fucose
modifying enzyme or GDP-fucose transport protein. Examples include
the host cells described in the following item 3.
[0345] The method for introducing RDO into various host cells
includes the methods for introducing recombinant vectors suitable
for various host cells described in the following ite 3.
[0346] The method for preparing cDNA encoding the GDP-fucose
synthase, the .alpha.1,6-fucose modifying enzyme or the GDP-fucose
transport protein includes the methods described in "Preparation of
DNA" in the item 1(1)(a) and the like.
[0347] The method for preparing a genomic DNA encoding the
GDP-fucose synthase, .alpha.1,6-fucose modifying enzyme or the
GDP-fucose transport protein includes the methods described in
"Preparation method of genomic DNA" in the item 1(1)(a) and the
like.
[0348] The nucleotide sequence of the DNA can be determined by
digesting it with appropriate restriction enzymes, cloning the
fragments into a plasmid such as pBluescript SK(-) (manufactured by
Stratagene), subjecting the clones to the reaction generally used
as a method for analyzing a nucleotide sequence such as the dideoxy
method of Sanger et al. [Proc. Natl. Acad. Sci. USA, 74, 5463
(1977)] or the like, and then analyzing the clones using an
automatic nucleotide sequence analyzer such as ABI PSISM 377DNA
Sequencer (manufactured by PE Biosystems) or the like.
[0349] The RDO can be prepared in the usual method or by using a
DNA synthesizer.
[0350] The method for selecting a cell in which a mutation
occurred, by introducing the RDO into the host cell, in the gene
encoding the targeting enzyme, the GDP-fucose synthase, the
.alpha.1,6-fucose modifying enzyme or the GDP-fucose transport
protein includes the methods for directly detecting mutations in
chromosomal genes described in Molecular Cloning, Second Edition,
Current Protocols in Molecular Biology and the like.
[0351] Furthermore, the method described in the item 1(1)(a) for
selecting a transformant based on the activity of the introduced
GDP-fucose synthase, .alpha.1,6-fucose modifying enzyme or
GDP-fucose transport protein and the method for selecting a
transformant based on the sugar chain structure of a glycoprotein
on the cell membrane described later in the item 1(5), and the
method for selecting a transformant based on the sugar structure of
a produced antibody molecule described later in the item 6 or 7 can
also be used.
[0352] The construct of the RDO can be designed in accordance with
the methods described in Science, 273, 1386 (1996); Nature
Medicine, 4, 285 (1998); Hepatology, 25, 1462 (1997); Gene Therapy,
5, 1960 (1999) J. Mol. Med., 75, 829 (1997); Proc. Natl. Acad. Sci.
USA, 96, 8774 (1999); Proc. Natl. Acad. Sci. USA, 96, 8768 (1999);
Nuc. Acids. Res., 27, 1323 (1999); Invest. Dematol., 111, 1172
(1998); Nature Biotech., 16, 1343 (1998); Nature Biotech., 18, 43
(2000); Nature Biotech., 18, 555 (2000); and the like.
[0353] (d) Preparation of Host Cell Used in the Method of the
Present Invention by RNAi Method
[0354] The host cell used in the method of the present invention
can be prepared by targeting a gene encoding the GDP-fucose
synthase, the .alpha.1,6-fucose modifying enzyme or the GDP-Fucose
transport protein according to the RNAi method, for example, as
follows.
[0355] A cDNA encoding the GDP-fucose synthase, the
.alpha.1,6-fucose modifying enzyme or the GDP-fucose transport
protein is prepared.
[0356] The nucleotide sequence of the prepared cDNA is
determined.
[0357] Based on the determined DNA sequence, an RNAi gene construct
of an appropriate length comprising a DNA encoding the GDP-fucose
synthase, the .alpha.1,6-fucose modifying enzyme or the GDP-fucose
transport protein or a DNA encoding a untranslated region, is
designed.
[0358] In order to express the RNAi gene in a cell, a recombinant
vector is prepared by inserting a fragment or full length of the
prepared DNA into downstream of the promoter of an appropriate
expression vector.
[0359] A transformant is obtained by introducing the recombinant
vector into a host cell suitable for the expression vector.
[0360] The host cell used in the method of the present invention
can be obtained by selecting a transformant based on the activity
of the introduced GDP-fucose synthase, .alpha.1,6-fucose modifying
enzyme or GDP-fucose transport protein, or the sugar chain
structure of the produced antibody molecule or of a glycoprotein on
the cell membrane.
[0361] As the host cell, any cell such as yeast, an animal cell, an
insect cell or a plant cell can be used, so long as it has a gene
encoding the target GDP-fucose synthase, .alpha.1,6-fucose
modifying enzyme or GDP-fucose transport protein. Examples include
the host cells described in the following item 3.
[0362] As the expression vector, a vector which is autonomously
replicable in the above host cell or can be integrated into the
chromosome and comprises a promoter at such a position that the
designed RNAi gene can be transferred is used. Examples include the
expression vectors described in the following item 3.
[0363] As the method for introducing a gene into various host
cells, the methods for introducing recombinant vectors suitable for
various host cells described in the following item 3 can be
used.
[0364] The method for selecting a transformant based on the
activity of the GDP-fucose synthase, the .alpha.1,6-fucose
modifying enzyme or the GDP-fucose transport protein includes the
methods described in the item 1(1)(a).
[0365] The method for selecting a transformant based on the sugar
chain structure of a glycoprotein on the cell membrane includes the
methods described in the following item 1(5). The method for
selecting a transformant based on the sugar chain structure of a
produced antibody molecule includes the methods described in the
following item 6 or 7.
[0366] The method for preparing cDNA of the GDP-fucose synthase,
the .alpha.1,6-fucose modifying enzyme or the GDP-fucose transport
protein includes the methods described in "Preparation of DNA" in
the item 1(1)(a) and the like.
[0367] In addition, the host cell used in the method of the present
invention can also be obtained without using an expression vector,
by directly introducing an RNAi gene designed based on the
nucleotide sequence of the GDP-fucose synthase, the
.alpha.1,6-fucose modifying enzyme or the GDP-fucose transport
protein.
[0368] The RNAi gene can be prepared in the usual method or by
using a DNA synthesizer.
[0369] The RNAi gene construct can be designed in accordance with
the methods described in Nature, 39, 806 (1998); Proc. Natl. Acad.
Sci. USA, 95, 15502 (1998); Nature, 395, 854 (1998); Proc. Natl.
Acad. Sci. USA, 96, 5049 (1999); Cell, 95, 1017 (1998); Proc. Natl.
Acad. Sci. USA 96, 1451 (1999); Proc. Natl. Acad. Sci. USA, 95,
13959 (1998), Nature Cell Biol., 2, 70 (2000), and the like.
[0370] (e) Preparation of Host Cell Used in the Method of the
Present Invention by Method Using Transposon
[0371] The host cell used in the method of the present invention
can be prepared by selecting a mutant based on the activity of the
GDP-fucose synthase, the .alpha.1,6-fucose modifying enzyme or the
GDP-fucose transport protein or the sugar chain structure of a
produced antibody molecule or of a glycoprotein on the cell
membrane by using a transposon system described in Nature Genet.,
25, 35 (2000) or the like.
[0372] The transposon system is a system in which a mutation is
induced by randomly inserting an exogenous gene into chromosome,
wherein an exogenous gene interposed between transposons is
generally used as a vector for inducing a mutation, and a
transposase expression vector for randomly inserting the gene into
chromosome is introduced into the cell at the same time.
[0373] Any transposase can be used, so long as it is suitable for
the sequence of the transposon to be used.
[0374] As the exogenous gene, any gene can be used, so long as it
can induce a mutation in the DNA of a host cell.
[0375] As the host cell, any cell such as yeast, an animal cell, an
insect cell or a plant cell can be used, so long as it has a gene
encoding the targeting GDP-fucose synthase, .alpha.1,6-fucose
modifying enzyme or GDP-fucose transport protein. Examples include
the host cells described in the following item 3. For introducing
the gene into various host cells, the method for introducing
recombinant vectors suitable for various host cells described in
the following item 3, can be used.
[0376] The method for selecting a mutant based on the activity of
the GDP-fucose synthase, the .alpha.1,6-fucose modifying enzyme or
the GDP-fucose transport protein includes the methods which will be
described above in the item 1(1)(a).
[0377] The method for selecting a mutant based on the sugar chain
structure of a glycoprotein on the cell membrane includes the
methods described in the following item 1(5). The method for
selecting a transformant based on the sugar chain structure of a
produced antibody molecule includes the methods described in the
following item 6 or 7.
[0378] (2) Method for Introducing Dominant Negative Mutant of
Enzyme
[0379] The host cell used in the method of the present invention
can be prepared by targeting a gene encoding the GDP-fucose
synthase, the .alpha.1,6-fucose modifying enzyme or the GDP-fucose
transport protein according to a technique for introducing a
dominant negative mutant of the enzyme. The GDP-fucose synthase
includes GMD, Fx, GFPP, fucokinase and the like. The
.alpha.1,6-fucose modifying enzyme includes
.alpha.1,6-fucosyltransferase, .alpha.-L-fucosidase and the like.
The GDP-fucose transport protein includes GDP-fucose transporter
and the like.
[0380] The enzymes catalyze specific reactions having substrate
specificity, and dominant negative mutants of the enzymes can be
prepared by disrupting the active center of the enzymes which have
the catalytic activity having substrate specificity. The method for
preparing a dominant negative mutant is specifically described as
follows with reference to GMD among the target enzymes.
[0381] As a result of the analysis of the three-dimensional
structure of E. coli-derived GMD, it has been found that 4 amino
acids (threonine at position 133, glutamic acid at position 135,
tyrosine at position 157 and lysine at position 161) have an
important function on the enzyme activity [Structure, 8, 2 (2000)].
That is, when mutants were prepared by substituting the 4 amino
acids with other different amino acids based on the
three-dimensional structure information, the enzyme activity of all
of the mutants was significantly decreased. On the other hand,
changes in the ability of GMD to bind to GMD coenzyme NADP and its
substrate GDP-mannose were hardly observed in the mutants.
Accordingly, a dominant negative mutant can be prepared by
substituting the 4 amino acids which control the enzyme activity of
GMD. A dominant negative mutant can be prepared by comparing the
homology and predicting the three-dimensional structure using the
amino acid sequence information based on the results of the E.
coli-derived GMD. For example, in GMD (SEQ ID NO:65) derived from
CHO cell, a dominant negative mutant can be prepared by
substituting threonine at position 155, glutamic acid at position
157, tyrosine at position 179 and lysine at position 183. Such a
gene into which amino acid substitution is introduced can be
prepared by the site-directed mutagenesis described in Molecular
Cloning, Second Edition, Current Protocols in Molecular Biology or
the like.
[0382] The host cell can be prepared by using the above prepared
dominant negative mutant gene of the target enzyme according to the
method described in Molecular Cloning, Second Edition, Current
Protocols in Molecular Biology, Manipulating the Mouse Embryo,
Second Edition or the like, for example, as follows.
[0383] A gene encoding the dominant negative mutant of the
GDP-fucose synthase, the .alpha.1,6-fucose modifying enzyme or the
GDP-fucose transport protein (hereinafter referred to as "dominant
negative mutant gene") is prepared.
[0384] Based on the full length DNA of the prepared dominant
negative mutant gene, a DNA fragment of an appropriate length
containing a DNA encoding the antibody molecule is prepared, if
necessary.
[0385] A recombinant vector is prepared by inserting the DNA
fragment or full length DNA into downstream of the promoter of an
appropriate expression vector.
[0386] A transformant is obtained by introducing the recombinant
vector into a host cell suitable for the expression vector.
[0387] The host cell used in the method of the present invention
can be prepared by selecting a transformant based on the activity
of the GDP-fucose synthase, the .alpha.1,6-fucose modifying enzyme
or the GDP-fucose transport protein, or the sugar chain structure
of a produced antibody molecule or of a glycoprotein on the cell
membrane.
[0388] As the host cell, any cell such as yeast, an animal cell, an
insect cell or a plant cell can be used, so long as it has a gene
encoding the GDP-fucose synthase, the .alpha.1,6-fucose modifying
enzyme or the GDP-fucose transport protein. Examples include the
host cells described in the following item 3.
[0389] As the expression vector, a vector which is autonomously
replicable in the host cell or can be integrated into the
chromosome and comprises a promoter at a position where
transcription of the DNA encoding the dominant negative mutant of
interest can be effected is used. Examples include the expression
vectors described in the following item 3.
[0390] For introducing the gene into various host cells, the method
for introducing recombinant vectors suitable for various host cells
described in the following item 3, can be used.
[0391] The method for selecting a mutant based on the activity of
the GDP-fucose synthase, the .alpha.1,6-fucose modifying enzyme or
the GDP-fucose transport protein includes the methods which will be
described in the above item 1(1)(a).
[0392] The method for selecting a mutant based on the sugar chain
structure of a glycoprotein on the cell membrane includes the
methods described in the following item 1(5). The method for
selecting a transformant based on the sugar chain structure of a
produced antibody molecule includes the methods described in the
following item 6 or 7.
[0393] (3) Method for Introducing Mutation into Enzyme
[0394] The host cell used in the method of the present invention
can be prepared by introducing a mutation into a gene encoding the
GDP-fucose synthase, the .alpha.1,6-fucose modifying enzyme or the
GDP-fucose transport protein, and then selecting a cell line of
interest in which the mutation occurred in the enzyme.
[0395] The GDP-fucose synthase includes GMD, Fx, GFPP, fucokinase
and the like. The .alpha.1,6-fucose modifying enzyme includes
.alpha.1,6-fucosyltransferase, .alpha.-L-fucosidase and the like.
The GDP-fucose transport protein includes GDP-fucose transporter
and the like.
[0396] The method for introducing mutation into an enzyme includes
1) a method in which a desired clone is selected from mutants
obtained by a mutation-inducing treatment of a parent cell line
with a mutagen or spontaneously generated mutants, based on the
activity of the GDP-fucose synthase, the .alpha.1,6-fucose
modifying enzyme or the GDP-fucose transport protein, 2) a method
in which a desired clone is selected from mutants obtained by a
mutation-inducing treatment of a parent cell line with a mutagen or
spontaneously generated mutants, based on the sugar chain structure
of a produced antibody molecule, 3) a method in which a desired
clone is selected from mutants obtained by a mutation-inducing
treatment of a parent cell line with a mutagen or spontaneously
generated mutants, based on the sugar chain structure of a
glycoprotein on the cell membrane, and the like.
[0397] As the mutation-inducing treatment, any treatment can be
used, so long as it can induce a point mutation or a deletion or
frame shift mutation in the DNA of cells of the parent cell
line.
[0398] Examples include treatment with ethyl nitrosourea,
nitrosoguanidine, benzopyrene or an acridine pigment and treatment
with radiation. Also, various alkylating agents and carcinogens can
be used as mutagens. The method for allowing a mutagen to act upon
cells includes the methods described in Tissue Culture Techniques,
3rd edition (Asakura Shoten), edited by Japanese Tissue Culture
Association (1996), Nature Genet., 24, 314 (2000) and the like.
[0399] The spontaneously generated mutant includes mutants which
are spontaneously formed by continuing subculture under general
cell culture conditions without applying special mutation-inducing
treatment.
[0400] The method for measuring the activity of the GDP-fucose
synthase, the .alpha.1,6-fucose modifying enzyme or the GDP-fucose
transport protein includes the methods described above in the item
1(1)(a). The method for distinguishing the sugar chain structure of
a produced antibody molecule includes the methods described in the
following item 6 or 7. The method for distinguishing the sugar
chain structure of a glycoprotein on the cell membrane includes the
methods described in the following item 1(5).
[0401] (4) Method for Suppressing Transcription and/or Translation
of Enzyme
[0402] The host cell used in the method of the present invention
can be prepared by targeting a gene encoding the GDP-fucose
synthase or the .alpha.1,6-fucose modifying enzyme or the
GDP-fucose transport protein and suppressing transcription and/or
translation of the target gene according to the antisense RNA/DNA
technique [Bioscience and Industry, 50, 322 (1992); Chemistry, 46,
681 (1991), Biotechnology, 9, 358 (1992), Trends in Biotechnology,
10, 87 (1992), Trends in Biotechnology, 10, 152 (1992), Cell
Engineering, 16, 1463 (1997)], the triple helix technique [Trends
in Biotechnology, 10, 132 (1992)] or the like.
[0403] The GDP-fucose synthase includes GMD, Fx, GFPP, fucokinase
and the like. The .alpha.1,6-fucose modifying enzyme includes
.alpha.1,6-fucosyltransferase, .alpha.-L-fucosidase and the like.
The GDP-fucose transport protein includes GDP-fucose transporter
and the like.
[0404] (5) Method for Selecting Clone Resistant to Lectin which
Recognizes Sugar Chain Structure in which 1-Position of Fucose is
Bound to 6-Position of N-acetylglucosamine in the Reducing End
through .alpha.-Bond in the N-glycoside-linked Sugar Chain
[0405] The host cell used in the method of the present invention
can be prepared by using a method for selecting a clone resistant
to a lectin which recognizes a sugar chain structure in which
1-position of fucose is bound to 6-position of N-acetylglucosamine
in the reducing end through .alpha.-bond in the N-glycoside-linked
sugar chain.
[0406] The method for selecting a clone resistant to a lectin which
recognizes a sugar chain structure in which 1-position of fucose is
bound to 6-position of N-acetylglucosamine in the reducing end
through .alpha.-bond in the N-glycoside-linked sugar chain includes
the methods using lectin described in Somatic Cell Mol. Genet., 12,
51 (1986) and the like.
[0407] As the lectin, any lectin can be used, so long as it is a
lectin which recognizes a sugar chain structure in which 1-position
of fucose is bound to 6-position of N-acetylglucosamine in the
reducing end through .alpha.-bond in the N-glycoside-linked sugar
chain. Examples include a Lens culinaris lectin LCA (lentil
agglutinin derived from Lens culinaris), a pea lectin PSA (pea
lectin derived from Pisum sativum), a broad bean lectin VFA
(agglutinin derived from Vicia faba), an Aleuria aurantia lectin
AAL (lectin derived from Aleuria aurantia) and the like.
[0408] Specifically, the clone of the present invention resistant
to a lectin which recognizes a sugar chain structure in which
1-position of fucose is bound to 6-position of N-acetylglucosamine
in the reducing end through (.alpha.-bond in the
NA-glycoside-linked sugar chain can be selected by culturing cells
by using a medium comprising the lectin at a concentration of 1
.mu.g/ml to 1 mg/ml for 1 day to 2 weeks, preferably 1 day to 1
week, subculturing surviving cells or picking up a colony and
transferring it into a culture vessel, and subsequently continuing
the culturing using the lectin-containing medium.
[0409] The method for confirming that the cell is a
lectin-resistant cell includes a method for confirming expression
of the GDP-fucose synthase, .alpha.1,6-fucose modifying enzyme or
the GDP-fucose transport protein, a method for culturing the cell
in a medium to which lectin is directly added. Specifically, when
the expression amount of the mRNA of .alpha.1,6-fucosyltransferase
which is one of .alpha.1,6-fucose modifying enzymes in the cell is
measured, a lectin-resistant cell decreases in an amount of the
mRNA expressed.
[0410] 2. Preparation of Transgenic Non-Human Animal or Plant or
the Progenies
[0411] The cell used in the method of the present invention can be
prepared by using a transgenic non-human animal or plant or the
progenies thereof in which a genomic gene is modified in such a
manner that the activity of the GDP-fucose synthase, the
.alpha.1,6-fucose modifying enzyme or the GDP-fucose transport
protein is decreased or deleted. The transgenic non-human animal or
plant or the progenies thereof can be prepared by targeting a gene
encoding the above protein according to the method similar to that
in the item 1.
[0412] In a transgenic non-human animal, the embryonic stem cell
used in the process of the present invention in which the activity
of the GDP-fucose synthase, the .alpha.1,6-fucose modifying enzyme
or the GDP-fucose transport protein is decreased or deleted can be
prepared applying the method similar to that in the item 1 to an
embryonic stem cell of the intended non-human animal such as
cattle, sheep, goat, pig, horse, mouse, rat, fowl, monkey or
rabbit.
[0413] Specifically, a mutant clone is prepared in which a gene
encoding the GDP-fucose synthase, the .alpha.1,6-fucose modifying
enzyme or the GDP-fucose transport protein is inactivated or
substituted with any nucleotide sequence, by a known homologous
recombination technique [e.g., Nature, 326, 6110, 295 (1987); Cell,
51, 3, 503 (1987); etc.]. Using the prepared mutant clone, a
chimeric individual comprising an embryonic stem cell clone and a
normal cell can be prepared by an injection chimera method into
blastocyst of fertilized egg of an animal or by an aggregation
chimera method. The chimeric individual is crossed with a normal
individual, so that a transgenic non-human animal in which the
activity of the GDP-fucose synthase, the .alpha.1,6-fucose
modifying enzyme or the GDP-fucose transport protein is decreased
or deleted in the whole body cells can be obtained.
[0414] The target vector for the homologous recombination of the
target gene can be prepared in accordance with a method described
in Gene Targeting, A Practical Approach, IRL Press at Oxford
University Press (1993); Preparation of Mutant Mice using ES Cells,
or the like. The target vector can be used as any of a replacement
type an insertion type and a gene trap type.
[0415] As the method for introducing the target vector into the
embryonic stem cell, any method can be used, so long as it can
introduce DNA into an animal cell. Examples include electroporation
[Cytotechnology, 3, 133 (1990)], the calcium phosphate method
(Japanese Published Unexamined Patent Application No. 227075/90),
the lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413
(1987)], the injection method [Manipulating the Mouse Embryo,
Second Edition], a method using particle gun (gene gun) (Japanese
Patent No. 2606856, Japanese Patent No. 2517813), the DEAE-dextran
method [Biomanual Series 4--Gene Transfer and Expression Analysis
(Yodo-sha), edited by Takashi Yokota and Kenichi Arai (1994)], the
virus vector method [Manipulating A Mouse Embryo, Second Edition]
and the like.
[0416] The method for efficiently selecting a homologous
recombinant includes a method such as the positive selection,
promoter selection, negative selection or poly A selection
described in Gene Targeting, A Practical Approach, IRL Press at
Oxford University Press (1993), Preparation of Mutant Mice using ES
Cells, or the like. Specifically, in the case of the target vector
containing hprt gene, it is introduced into the hprt gene-defected
embryonic stem cell, the embryonic stem cell is cultured in a
medium containing aminopterin, hypoxanthine and thymidine, and
positive selection which selects the homologous recombinant of the
hprt gene can be carried out by selecting an a homogenous
recombinant containing an aminopterin-resistant clone. In the case
of the target vector containing a neomycin-resistant gene, the
vector-introduced embryonic stem cell is cultured in a medium
containing G418, and positive selection which selects a homogenous
recombinant containing a neomycin-resistant gene can be carried out
by selecting a G418-resistant clone. In the case of the target
vector containing DT gene, the vector-introduced embryonic stem
cell is cultured, and negative selection selecting a DT gene-free
homogenous recombinant can be carried out by selecting the grown
clone (in the recombinants introduced into a chromosome at random
rather than the homogenous recombination, since the DT gene is
expressed while integrated in the chromosome, the recombinants
cannot grow because of the toxicity of DT). The method for
selecting the homogenous recombinant of interest among the selected
clones include the Southern hybridization for genomic DNA
(Molecular Cloning, Second Edition), PCR [PCR Protocols, Academic
Press (1990)] and the like.
[0417] When the embryonic stem cell is introduced into a fertilized
egg by using an aggregation chimera method, in general, a
fertilized egg at the development stage before 8-cell stage is
preferably used. When the embryonic stem cell is introduced into a
fertilized egg by using an injection chimera method, in general, it
is preferred that a fertilized egg at the development stage from
8-cell stage to blastocyst stage is preferably used.
[0418] When the fertilized egg is transplanted into a female mouse,
it is preferred that a fertilized egg obtained from a
pseudopregnant female mouse in which fertility is induced by mating
with a male non-human mammal which is subjected to vasoligation is
artificially transplanted or implanted. Although the psuedopregnant
female mouse can be obtained by natural mating, the pseudopregnant
female mouse in which fertility is induced can be obtained by
mating with a male mouse after administration of a luteinizing
hormone-releasing hormone (hereinafter referred to as "LHRH") or
its analogue thereof. The analogue of LHRH includes
[3,5-Dil-Tyr5]-LHRH, [Gln8]-LHRH, [D-Ala6]-LHRH,
des-Gly10-[D-His(Bzl)6]-- LHRH ethylamide and the like.
[0419] Also, a fertilized egg cell of the present invention in
which the activity of the GDP-fucose synthase, the
.alpha.1,6-fucose modifying enzyme or the GDP-fucose transport
protein is decreased or deleted can be prepared by applying the
method similar to that in the item 1 to fertilized egg of a
non-human animal of interest such as cattle, sheep, goat, pig,
horse, mouse, rat, fowl, monkey, rabbit or the like.
[0420] A transgenic non-human animal. An which the activity of the
GDP-fucose synthase, the .alpha.1,6-fucose modifying enzyme or the
GDP-fucose transport protein is decreased or deleted can be
prepared by transplanting the prepared fertilized egg cell into the
oviduct or uterus of a pseudopregnant female using the embryo
transplantation method described in Manipulating Mouse Embryo,
Second Edition or the like, followed by childbirth by the
animal.
[0421] In a transgenic plant, the callus of the present invention
in which the activity of the GDP-fucose synthase or the activity of
an enzyme relating to modification of a sugar chain in which
1-position of fucose is bound to 6- or 3-position of
N-acetylglucosamine in the reducing end in the N-glycoside-linked
complex sugar chain is decreased or deleted can be prepared by
applying the method similar to that in the item 1 to a callus or
cell of the plant of interest.
[0422] A transgenic plant in which the activity of the GDP-fucose
synthase, the .alpha.1,6-fucose modifying enzyme or the GDP-fucose
transport protein is decreased or deleted can be prepared by
culturing the prepared callus in a medium comprising auxin and
cytokinin to redifferentiate it in accordance with conventional
methods [Tissue Culture (Soshiki Baiyo), 20 (1994); Tissue Culture
(Soshiki Baiyo)e, 21 (1995); Trends in Biotechnology, 15, 45
(1997)].
[0423] 3. Method for Producing Antibody Composition
[0424] The antibody composition can be obtained by expressing it in
a host cell by using the methods described in Molecular Cloning,
Second Edition; Current Protocols in Molecular Biology; Antibodies,
A Laboratory Manual, Cold Spring Harbor Laboratory, 1988
(hereinafter sometimes referred to as "Antibodies"); Monoclonal
Antibodies: Principles and Practice, Third Edition, Acad. Press,
1996 (hereinafter sometimes referred to as "Monoclonal
Antibodies"); and Antibody Engineering, A Practical Approach, IRL
Press at Oxford University Press, 1996 (hereinafter sometimes
referred to as "Antibody Engineering"), for example, as
follows.
[0425] A full length cDNA of an antibody molecule is prepared, and
a DNA fragment of an appropriate length comprising a DNA encoding
the antibody molecule is prepared.
[0426] A recombinant vector is prepared by inserting the DNA
fragment or the full length cDNA into downstream of the promoter of
an appropriate expression vector.
[0427] A transformant which produces the antibody molecule can be
obtained by introducing the recombinant vector into a host cell
suitable for the expression vector.
[0428] As the host cell, the host cell of any cell such as yeast,
an animal cell, an insect cell, a plant cell or the like can be
used, so long as it can express the gene of interest.
[0429] A cell such as yeast, an animal cell, an insect cell, a
plant cell or the like into which an enzyme re lating to the
modification of an N-glycoside-linked sugar chain which binds to
the Fc region of the antibody molecule is introduced by a genetic
engineering technique can also be used as the host cell.
[0430] As the expression vector, a vector which is autonomously
replicable in the host cell or can be integrated into the
chromosome and comprises a promoter at such a position that the DNA
encoding the antibody molecule of interest can be transferred is
used.
[0431] The cDNA can be prepared from a human or non-human tissue or
cell by using a probe primer specific for the antibody molecule of
interest and the like according to the methods described in
"Preparation of DNA" in the item 1(1)(a).
[0432] When yeast is used as the host cell, the expression vector
includes YEP13 (ATCC 37115), YEp24 (ATCC 37051), YCp50 (ATCC 37419)
and the like.
[0433] As the promoter, any promoter can be used so long as it can
function in yeast. Examples include a promoter of a gene relating
to the glycolytic pathway such as a hexose kinase, PHOS promoter,
PGK promoter, GAP promoter, ADH promoter, gal 1 promoter, gal 10
promoter, heat shock protein promoter, MF.alpha.1 promoter, CUP 1
promoter and the like.
[0434] The host cell includes yeast belonging to the genus
Saccharomyces, the genus Schizosaccharomyces, the genus
Kluyveromyces, the genus Trichosporon, the genus Schwanniomyces and
the like, such as Saccharomyces cerevisiae, Schizosaccharomyces
pombe, Kluyveromyces lactis, Trichosporon pullulans and
Schwanniomyces alluvius.
[0435] As the method for introducing the recombinant vector, any
method can be used, so long as it can introduce DNA into yeast.
Examples include electroporation [Methods in Enzymology, 194, 182
(1990)], spheroplast method [Proc Natl. Acad. Sci. USA, 84, 1929
(1978)], lithium acetate method [J. Bacteriol., 153, 163 (1983)], a
method described in Proc. Natl. Acad. Sci. USA, 75, 1929 (1978) and
the like.
[0436] When an animal cell is used as the host cell, the expression
vector includes pcDNAI, pcDM8 (available from Funakoshi), pAGE107
[Japanese Published Unexamined Patent Application No. 22979/91,
Cytotechnology, 3, 133 (1990)], pAS3-3 (Japanese Published
Unexamined Patent Application No. 227075/90), pCDM8 [Nature, 329,
840 (1987)], pcDNAI/Amp (manufactured by Invitrogen), pREP4
(manufactured by Invitrogen), pAGE103 [J. Biochemistry, 101, 1307
(1987)], pAGE210 and the like.
[0437] As the promoter, any promoter can be used, so long as it can
function in an animal cell. Examples include a promoter of IE
(immediate early) gene of cytomegalovirus (CMV), an early promoter
of SV40, a promoter of retrovirus, a promoter of metallothionein, a
heat shock promoter, an SR.alpha. promoter and the like. Also, an
enhancer of the IE gene of human CMV may be used together with the
promoter.
[0438] The host cell includes a human cell such as Namalwa cell, a
monkey cell such as COS cell, a Chinese hamster cell such as CHO
cell or HBT5637 (Japanese Published Unexamined Patent Application
No. 299/88), a rat myeloma cell, a mouse myeloma cell, a cell
derived from Syrian hamster kidney, an embryonic stem cell, a
fertilized egg cell and the like.
[0439] As the method for introducing the recombinant vector, any
method can be used, so long as it can introduce DNA into an animal
cell. Examples include electroporation [Cytotechnology, 3, 133
(1990)], the calcium phosphate method (Japanese Published
Unexamined Patent Application No. 227075/90), the lipofection
method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)], the injection
method [Manipulating the Mouse Embryo, A Laboratory Manual], a
method using particle gun (gene gun) (Japanese Patent No. 2606856,
Japanese Patent No. 2517813), the DEAE-dextran method [Biomanual
Series 4--Gene Transfer and Expression Analysis (Yodo-sha), edited
by Takashi Yokota and Kenichi Arai (1994)], the virus vector method
[Manipulating Mouse Embryo, Second Edition] and the like.
[0440] When an insect cell is used as the host cell, the protein
can be expressed by the method described in Current Protocols in
Molecular Biology, Baculovirus Expression Vectors, A Laboratory
Manual, W.H. Freeman and Company, New York (1992), Bio/Technology,
6, 47 (1988) or the like.
[0441] That is, the protein can be expressed by co-introducing a
recombinant gene-introducing vector and a baculovirus into an
insect cell to obtain a recombinant virus in an insect cell culture
supernatant and then infecting the insect cell with the recombinant
virus.
[0442] The gene introducing vector used in the method includes
pVL1392, pVL1393, pBlueBacIII (all manufactured by Invitrogen) and
the like.
[0443] The baculovirus includes Autographa californica nuclear
polyhedrosis virus which is infected by an insect of the family
Barathra.
[0444] The insect cell includes Spodoptera frugiperda oocytes Sf9
and Sf21 [Current Protocols in Molecular Biology, Baculovirus
Expression Vectors, A Laboratory Manual, W.H. Freeman and Company,
New York (1992)], a Trichoplusia ni oocyte High 5 (manufactured by
Invitrogen) and the like.
[0445] The method for co-introducing the above recombinant
gene-introducing vector and the baculovirus to the insect cells for
preparing the above recombinant virus includes the calcium
phosphate method (Japanese Published Unexamined Patent Application
No. 227075/90), the lipofection method [Proc. Natl. Acad. Sci. USA,
84, 7413 (1987)] and the like.
[0446] When a plant cell is used as the host cell, the expression
vector includes Ti plasmid, tobacco mosaic virus and the like.
[0447] As the promoter, any promoter can be used, so long as it can
function in a plant cell. Examples include cauliflower mosaic virus
(CaMV) 35S promoter, rice actin 1 promoter and the like.
[0448] The host cell includes plant cells of tobacco, potato,
tomato, carrot, soybean, rape, alfalfa, rice, wheat, barley and the
like.
[0449] As the method for introducing the recombinant vector, any
method can be used, so long as it can introduce DNA into a plant
cell. Examples include a method using Agrobacterium (Japanese
Published Unexamined Patent Application No. 140885/84, Japanese
Published Unexamined Patent Application No. 70080/85, WO94/00977),
electroporation (Japanese Published Unexamined Patent Application
No. 251887/85), a method using a particle gun (gene gun) (Japanese
Patent No. 2606856, Japanese Patent No. 2517913) and the like.
[0450] As the method for expressing an antibody gene, secretion
production, expression of a fusion protein of the Fc region with
other protein and the like can be carried out in accordance with
the method described in Molecular Cloning, Second Edition or the
like, in addition to the direct expression.
[0451] When a gene is expressed by a microorganism, yeast, an
animal cell, an insect cell or a plant cell into which a gene
relating to the synthesis of a sugar chain is introduced, an
antibody molecule to which a sugar or a sugar chain is added by the
introduced gene can be obtained.
[0452] An antibody composition can be produced by culturing the
obtained transformant in a medium to produce and accumulate the
antibody molecule in the culture and then recovering it from the
resulting culture. The method for culturing the transformant in a
medium can be carried out in accordance with a general method which
is used for the culturing of host cells.
[0453] As the medium for culturing a transformant obtained using
yeast as the host cell, the medium may be either a natural medium
or a synthetic medium, so long as it comprises materials such as a
carbon source, a nitrogen source and an inorganic salt which can be
assimilated by the organism and culturing of the transformant can
be efficiently carried out.
[0454] As the carbon source, those which can be assimilated by the
organism can be used. Examples include carbohydrates such as
glucose, fructose, sucrose, molasses thereof, starch and starch
hydrolysate; organic acids such as acetic acid and propionic acid,
alcohols such as ethanol and propanol; and the like.
[0455] The nitrogen source includes ammonia; ammonium salts of
inorganic acid or organic acid such as ammonium chloride, ammonium
sulfate, ammonium acetate and ammonium phosphate; other
nitrogen-containing compounds; peptone; meat extract; yeast
extract; corn steep liquor; casein hydrolysate; soybean meal;
soybean meal hydrolysate; various fermented cells and hydrolysates
thereof; and the like.
[0456] The inorganic salt includes potassium dihydrogen phosphate,
dipotassium hydrogen phosphate, magnesium phosphate, magnesium
sulfate, sodium chloride, ferrous sulfate, manganese sulfate,
copper sulfate, calcium carbonate, and the like.
[0457] The culturing is carried out generally under aerobic
conditions such as a shaking culture or submerged-aeration stirring
culture. The culturing temperature is preferably at 15 to
40.degree. C., and the culturing time is generally 16 hours to 7
days. During the culturing, the pH is maintained at 3.0 to 9.0. The
pH is adjusted using an inorganic or organic acid, an alkali
solution, urea, calcium carbonate, ammonia or the like.
[0458] Furthermore, if necessary, an antibiotic such as ampicillin
or tetracycline can be added to the medium during the
culturing.
[0459] When yeast transformed with a recombinant vector obtained by
using an inducible promoter as the promoter is cultured, an inducer
can be added to the medium, if necessary. For example, when yeast
transformed with a recombinant vector obtained by using lac
promoter is cultured, isopropyl-.beta.-D-thiogalactopyranoside and
the like can be added to the medium, and when yeast transformed
with a recombinant vector obtained by using trp promoter is
cultured, indoleacrylic acid and the like can be added to the
medium.
[0460] When a transformant obtained by using an animal cell as the
host is cultured, the medium includes generally used RPMI 1640
medium [The Journal of the American Medical Association, 199, 519
(1967)], Eagle's MEM medium [Science 122, 501 (1952)], Dulbecco's
modified MEM medium [Virology, 8, 396 (1959)], 199 medium
[Proceeding of the Society for the Biological Medicine 73, 1
(1950)] and Whitten's medium [Developmental Engineering
Experimentation Manual--Preparation of Transgenic Mice (Kodan-sha),
edited by M. Katsuki (1987)], the media to which fetal calf serum,
etc. a re added, and the like.
[0461] The culturing is carried out generally at conditions under
pH of 6 to 8 and 30 to 40.degree. C. for 1 to 7 days in the
presence of 5% CO.sub.2 and the like, for 1 to 7 days.
[0462] Furthermore, if necessary, an antibiotic such as kanamycin
or penicillin can be added to the medium during the culturing.
[0463] The medium for culturing a transformant obtained by using an
insect cell as the host includes generally used TNM-FH medium
(manufactured by Pharmingen), Sf-900 II SFM medium (manufactured by
Life Technologies), ExCell 400 and ExCell 405 (both manufactured by
JRH Biosciences), Grace's Insect Medium [Nature, 195, 788 (1962))
and the like.
[0464] The culturing is carried out generally at conditions under
pH of 6 to 7 and 25 to 30.degree. C. and the like, for 1 to 5
days.
[0465] Furthermore, if necessary, an antibiotic such as gentamicin
can be added to the medium during the culturing.
[0466] A transformant obtained by using a plant cell as the host
can be cultured as a cell or by differentiating it into a plant
cell or organ. The medium for culturing the transformant includes
generally used Murashige and Skoog (MS) medium and White medium,
the media to which a plant hormone such as auxin or cytokinin is
added, and the like.
[0467] The culturing is carried out generally at conditions under
pH of 5 to 9 and 20 to 40.degree. C. for 3 to 60 days.
[0468] Furthermore, if necessary, an antibiotic such as kanamycin
or hygromycin can be added to the medium during the culturing.
[0469] As described above, an antibody composition can be produced
by culturing a transformant derived from yeast, an animal cell, an
insect cell or a plant cell, which comprises a recombinant vector
into which a DNA encoding an antibody molecule is inserted, in
accordance with a general culturing method, to thereby produce and
accumulate the antibody composition, and then recovering the
antibody composition from the culture.
[0470] As the method for expressing the gene encoding an antibody,
secretion production, expression of a fusion protein and the like
can be carried out in accordance with the method described in
Molecular Cloning, Second Edition in addition to the direct
expression.
[0471] The method for producing an antibody composition includes a
method of intracellular expression in a host cell, a method of
extracellular secretion from a host cell, and a method of
production on a host cell membrane outer envelope. The method can
be selected by changing the host cell used or the structure of the
antibody composition produced.
[0472] The method for producing an antibody composition includes a
method of intracellular expression in a host cell, a method of
extracellular secretion from a host cell, and a method of
production on a host cell membrane outer envelope. The method can
be selected by changing the host cell used or the structure of the
antibody composition produced.
[0473] When the antibody composition is produced in a host cell or
on a host cell membrane outer envelope, it can be positively
secreted extracellularly in accordance with the method of Paulson
et al. J. Biol. Chem., 264, 17619 (1989)], the method of Lowe ea
al. [Proc. Natl. Acad. Sci. USA, 86, 8227 (1989), Genes Develop.,
4, 1288 (1990)], the methods described in Japanese Published
Unexamined Patent Application No. 336963/93 and Japanese Published
Unexamined Patent Application No. 823021/94 and the like.
[0474] That is, an antibody molecule of interest can be positively
secreted extracellularly from a host cell by inserting a DNA
encoding the antibody molecule and a DNA encoding a signal peptide
suitable for the expression of the antibody molecule into an
expression vector according to a gene recombination technique and
then expressing the antibody molecule.
[0475] Also, its production amount can be increased in accordance
with the method described in Japanese Published Unexamined Patent
Application No. 227075/90 according to a gene amplification system
using a dihydrofolate reductase gene.
[0476] In addition, the antibody composition can also be produced
by using a gene-introduced animal individual (transgenic non-human
animal) or a plant individual (transgenic plant) which is
constructed by the redifferentiation of an animal or plant cell
into which the gene is introduced.
[0477] When the transformant is an animal individual or a plant
individual, an antibody composition can be produced in accordance
with a general method by rearing or cultivating it to thereby
produce and accumulate the antibody composition and then recovering
the antibody composition from the animal or plant-individual.
[0478] The method for producing an antibody composition using an
animal individual includes a method in which the antibody
composition of interest is produced in an animal constructed by
introducing a gene in accordance with a known method [American
Journal of Clinical Nutrition, 63, 639S (1996); American Journal of
Clinical Nutrition, 63, 627S (1996), Bio/Technology, 9, 830
(1991)].
[0479] In the case of an animal individual, an antibody composition
can be produced, for example, by rearing a transgenic non-human
animal into which a DNA encoding an antibody molecule is introduced
to thereby produce and accumulate the antibody composition in the
animal, and then recovering the antibody composition from the
animal. The place of the animal where the composition is produced
and accumulated includes milk (Japanese Published Unexamined Patent
Application No. 309192/88) and eggs of the animal. As the promoter
used in this case, any promoter can be used, so long as it can
function in an animal. Preferred examples include mammary gland
cell-specific promoters such as .alpha. casein promoter, .beta.
casein promoter, .beta. lactoglobulin promoter, whey acidic protein
promoter and the like.
[0480] The method for producing an antibody composition using a
plant individual includes a method in which an antibody composition
is produced by cultivating a transgenic plant into which a DNA
encoding an antibody molecule is introduced by a known method
[Tissue Culture (Soshiki Baiyo), 20 (1994); Tissue Culture (Soshiki
Baiyo), 21 (1995); Trends in Biotechnology, 15, 45 (1997)] to
produce and accumulate the antibody composition in the plant, and
then recovering the antibody composition from the plant.
[0481] Regarding purification of an antibody composition produced
by a transformant into which a gene encoding an antibody molecule
is introduced, for example, when the antibody composition is
intracellularly expressed in a dissolved state, the cells after
culturing are recovered by centrifugation, suspended in an aqueous
buffer and then disrupted by using ultrasonic oscillator, French
press, Manton Gaulin homogenizer, dynomill or the like to obtain a
cell-free extract. A purified product of the antibody composition
can be obtained from a supernatant obtained by centrifuging the
cell-free extract according to a general enzyme isolation
purification techniques such as solvent extraction; salting out or
desalting with ammonium sulfate; precipitation with an organic
solvent; anion exchange chromatography using a resin such as
diethylaminoethyl (DEAE)-Sepharose or DIAION HPA-75 (manufactured
by Mitsubishi Chemical); cation exchange chromatography using a
resin such as S-Sepharose FF (manufactured by Pharmacia),
hydrophobic chromatography using a resin such as butyl-Sepharose or
phenyl-Sepharose, gel filtration using a molecular sieve; affinity
chromatography, chromatofocusing; electrophoresis such as
isoelectric focusing, and the like which may be used alone or in
combination.
[0482] Also, when the antibody composition is expressed
intracellularly by forming an insoluble body, the cells are
recovered, disrupted and centrifuged in the same manner, and the
insoluble body of the antibody composition is recovered as a
precipitation fraction. The recovered insoluble body of the
antibody composition is solubilized by using a protein denaturing
agent. The antibody composition is made into a normal
three-dimensional structure by diluting or dialyzing the
solubilized solution, and then a purified product of the antibody
composition is obtained by the same isolation purification
method.
[0483] When the antibody composition is secreted extracellularly,
the antibody composition or derivatives thereof can be recovered
from the culture supernatant. That is, the culture is treated
according to a technique such as centrifugation as described above
to obtain a soluble fraction, and a purified preparation of the
antibody composition can be obtained from the soluble fraction by
the same isolation purification method as described above.
[0484] The thus obtained antibody composition includes an antibody,
the fragment of the antibody, a fusion protein comprising the Fc
region of the antibody, and the like.
[0485] As an example for obtaining the antibody composition, a
method for producing a composition of a humanized antibody and Fc
fusion protein is described below in detail, but other antibody
compositions can also be obtained in a manner similar to the
method.
[0486] A. Preparation of Humanized Antibody Composition
[0487] (1) Construction of Vector for Expression of Humanized
Antibody
[0488] A vector for expression of humanized antibody is an
expression vector for animal cell into which genes encoding CH and
CL of a human antibody are inserted, which can be constructed by
cloning each of genes encoding CH and CL of a human antibody into
an expression vector for animal cell.
[0489] The C regions of a human antibody may be CH and CL of any
human antibody. Examples include the C region belonging to IgG1
subclass in the H chain of a human antibody (hereinafter referred
to as "hC.gamma.1"), the C region belonging to .kappa. class in the
L chain of a human antibody (hereinafter referred to as
"hC.kappa."), and the like.
[0490] As the genes encoding CH and CL of a human antibody, a
chromosomal DNA comprising an exon and an intron can be used, and a
cDNA can also be used.
[0491] As the expression vector for animal cell, any vector can be,
used, so long as a gene encoding the C region of a human antibody
can be inserted thereinto and expressed therein. Examples include
pAGE107 [Cytotechnology, 3, 133 (1990)], pAGE103 [J. Biochem., 101,
1307 (1987)], pHSG274 [Gene, 27, 223 (1984)], pKCR [Proc. Natl.
Acad. Sci. USA, 78, 1527 (1981), pSG1 .beta. d2-4 [Cytotechnology,
4, 173 (1990)] and the like. The promoter and enhancer in the
expression vector for animal cell includes SV40 early promoter and
enhancer [J. Biochem., 101, 1307 (1987)], Moloney mouse leukemia
virus LTR promoter [Biochem. Biophys. Res. Commun, 149, 960
(1987)], immunoglobulin H chain promoter [Cell, 41, 479 (1985)] and
enhancer [Cell, 33, 717 (1983)], and the like.
[0492] The vector for expression of humanized antibody may be
either of a type in which genes encoding the H chain and L chain of
an antibody exist on separate vectors or of a type in which both
genes exist on the same vector (hereinafter referred to "tandem
type"). In respect of easiness of construction of a vector for
expression of humanized antibody, easiness of introduction into
animal cells, and balance between the expression amounts of the H
and L chains consisting of an antibody in animal cells, a tandem
type of the vector for humanized antibody expression is more
preferred [J. Immunol. Methods, 167, 271 (1994)].
[0493] The constructed vector for expression of humanized antibody
can be used for expression of a human chimeric antibody and a human
CDR-grafted antibody in animal cells.
[0494] (2) Preparation Method of cDNA Encoding V Region of
Non-Human Animal Antibody
[0495] cDNAs encoding VH and VL of a non-human animal antibody such
as mouse antibody can be obtained in the following manner.
[0496] A cDNA is synthesized from mRNA extracted from a hybridoma
cell which produces the mouse antibody of interest. The synthesized
cDNA is cloned into a vector such as a phage or a plasmid to obtain
a cDNA library. Each of a recombinant phage or recombinant plasmid
comprising a cDNA encoding VH and a recombinant phage or
recombinant plasmid comprising a cDNA encoding VL is isolated from
the library by using a C region part or a V region part of an
existing mouse antibody as the probe. Full nucleotide sequences of
VH and VL of the mouse antibody of interest on the recombinant
phage or recombinant plasmid are determined, and full length amino
acid sequences of VH and VL are deduced from the nucleotide
sequences.
[0497] As the non-human animal, any animal such as mouse, rat,
hamster or rabbit can be used, so long as a hybridoma cell can be
produced therefrom.
[0498] The method for preparing a total RNA from a hybridoma cell
includes the guanidine thiocyanate-cesium trifluoroacetate method
[Methods in Enzymology, 154, 3 (1987)] and the like, and the method
for preparing mRNA from total RNA includes an oligo(dT)-immobilized
cellulose column method (Molecular Cloning, Second Edition) and the
like. In addition, a kit for preparing mRNA from a hybridoma cell
includes Fast Track mRNA Isolation Kit (manufactured by
Invitrogen), Quick Prep mRNA Purification Kit (manufactured by
Pharmacia) and the like.
[0499] The method for synthesizing a cDNA and preparing a cDNA
library includes the usual methods (Molecular Cloning, Second
Edition, Current Protocols in Molecular Biology, Supplement 1-34),
methods using a commercially available kit such as SuperScript.TM.,
Plasmid System for cDNA Synthesis and Plasmid Cloning (manufactured
by GIBCO BRL) or ZAP-cDNA Synthesis Kit (manufactured by
Stratagene), and the like.
[0500] In preparing the cDNA library, the vector into which a cDNA
synthesized by using mRNA extracted from a hybridoma cell as the
template is inserted may be any vector, so long as the cDNA can be
inserted. Examples include ZAP Express [Strategies, 5, 58 (1992)],
pBluescript II SK(+) [Nucleic Acids Research, 17, 9494 (1989)],
.lambda.zapII (manufactured by Stratagene), .lambda.gt10 and
.lambda.gt11 [DNA Cloning, A Practical Approach, I, 49 (1985)],
Lambda BlueMid (manufactured by Clontech), .lambda.ExCell, pT7T318U
(manufactured by Pharmacia), pcD2 [Mol. Cell. Biol., 3, 280
(1983)], pUC18 [Gene, 33, 103 (1985)] and the like.
[0501] As Escherichia coli into which the cDNA library constructed
from a phage or plasmid vector is introduced, any Escherichia coli
can be used, so long as the cDNA library can be introduced,
expressed and maintained. Examples include XL1-Blue MRF'
[Strategies, 5, 81 (1992)], C600 [Genetics, 39, 440 (1954)], Y1088
and Y1090 [Science, 222, 778 (1983)], NM522 [J. Mol. Biol., 166, 1
(1983)], K802 [J. Mol. Biol., 16, 118 (1966)], JM105 [Gene, 38, 275
(1985)] and the like.
[0502] As the method for selecting a cDNA clone encoding VH and VL
of a non-human animal antibody from the cDNA library, a colony
hybridization or a plaque hybridization using an isotope- or
fluorescence-labeled probe can be used (Molecular Cloning, Second
Edition). The cDNA encoding VH and VL can also be prepared by
preparing primers and carrying out polymerase chain reaction
(hereinafter referred to as "PCR"; Molecular Cloning, Second
Edition Current Protocols in Molecular Biology, Supplement 1-34)
using a cDNA synthesized from mRNA or a cDNA library as the
template.
[0503] The nucleotide sequences of the cDNAs can be determined by
digesting the selected cDNAs with appropriate restriction enzymes,
cloning the fragments into a plasmid such as pBluescript SK(-)
(manufactured by Stratagene), carrying out the reaction of a
generally used nucleotide sequence analyzing method such as the
dideoxy method of Sanger el al. [Proc. Natl. Acad. Sci. USA, 74,
5463 (1977)], and then analyzing the clones using an automatic
nucleotide sequence analyzer such as A.L.F. DNA Sequencer
(manufactured by Pharmacia).
[0504] Whether or not the obtained cDNAs encode the full length
amino acid sequences of VH and VL of the antibody comprising a
secretory signal sequence can be confirmed by deducing the full
length amino acid sequences of VH and V from the determined
nucleotide sequence and comparing them with the full length amino
acid sequences of VH and VL of known antibodies [Sequences of
Proteins of Immunological Interest, US Dep. Health and Human
Services (1991), hereinafter referred to as "Sequences of Proteins
of Immunological Interest"].
[0505] (3) Analysis of Amino Acid Sequence of V Region of Non-Human
Animal Antibody
[0506] Regarding the full length amino acid sequences of VH and VL
of the antibody comprising a secretory signal sequence, the length
of the secretory signal sequence and the N-terminal amino acid
sequences can be deduced and subgroups to which they belong can
also be found, by comparing them with the full length amino acid
sequences of VH and VL of known antibodies (Sequences of Proteins
of Immunological Interest). In addition, the amino acid sequences
of each CDR of VH and VL can also be found by comparing them with
the amino acid sequences of VH and VL of known antibodies
(Sequences of Proteins of Immunological Interest).
[0507] (4) Construction of Human Chimeric Antibody Expression
Vector
[0508] A human chimeric antibody expression vector can be
constructed by cloning 4 cDNAs encoding VH and VL of a non-human
animal antibody into upstream of genes encoding CH and CL of a
human antibody in the vector for expression of humanized antibody
constructed described in the item 3(1). For example, a human
chimeric antibody expression vector can be constructed by linking
each of cDNAs encoding VH and VL of a non-human animal antibody to
a synthetic DNA comprising nucleotide sequences at the 3'-terminals
of VH and VL of a non-human animal antibody and nucleotide
sequences at the 5'-terminals of CH and CL of a human antibody and
also having a recognition sequence of an appropriate restriction
enzyme at both terminals, and by cloning them into upstream of
genes encoding CH and CL of a human antibody contained in the
vector for expression of humanized antibody constructed described
in the item 3(1) in such a manner that they can be expressed in a
suitable form.
[0509] (5) Construction of cDNA Encoding V Region of Human
CDR-Grafted Antibody
[0510] cDNAs encoding VH and VL of a human CDR-grafted antibody can
be obtained as follows. First, amino acid sequences of the
frameworks (hereinafter referred to as "FR") of VH and VL of a
human antibody for grafting CDR of VH and VL of a non-human animal
antibody is selected. As the amino acid sequences of FRs of VH and
VL of a human antibody, any amino acid sequences can be used so
long as they are derived from a human antibody. Examples include
amino acid sequences of FRs of VH and VL of human antibodies
registered at databases such as Protein Data Bank, amino acid
sequences common in each subgroup of FRs of VH and VL of human
antibodies (Sequences of Proteins of Immunological Interest) and
the like. In order to produce a human CDR-grafted antibody having
enough activities, it is preferred to select an amino acid sequence
having homology as high as possible (at least 60% or more) with
amino acid sequences of VH and VL of a non-human animal antibody of
interest.
[0511] Next, the amino acid sequences of CDRs of VH and VL of the
non-human animal antibody of interest are grafted to the selected
amino acid sequences of FRs of VH and VL of a human antibody to
design amino acid sequences of VH and VL of the human CDR-grafted
antibody. The designed amino acid sequences are converted into DNA
sequences by considering the frequency of codon usage found in
nucleotide sequences of antibody genes (Sequences of Proteins of
Immunological Interest), and the DNA sequences encoding the amino
acid sequences of VH and VL of the human CDR-grafted antibody are
designed. Based on the designed DNA sequences, several synthetic
DNAs having a length of about 100 bases are synthesized, and PCR is
carried out by using them. In this case, it is preferred in each of
the H chain and the L chain that 6 synthetic DNAs are designed in
view of the reaction efficiency of PCR and the lengths of DNAs
which can be synthesized.
[0512] Also, they can be easily cloned into the vector for
expression of humanized antibody described in the item 3(1) by
introducing recognition sequences of an appropriate restriction
enzyme into the 5'-terminals of the synthetic DNA on both
terminals. After the PCR, the amplified product is cloned into a
plasmid such as pBluescript SK(-) (manufactured by Stratagene) and
the nucleotide sequences are determined by the method in the item
3(2) to thereby obtain a plasmid having DNA sequences encoding the
amino acid sequences of VH and VL of the desired human CDR-grafted
antibody.
[0513] (6) Construction of Human CDR-Grafted Antibody Expression
Vector
[0514] A human CDR-grafted antibody expression vector can be
constructed by cloning the cDNAs encoding VH and VL of the human
CDR-grafted antibody constructed in the item 3(5) into upstream of
the gene encoding CH and CL of a human antibody in the vector for
expression of humanized antibody described in the item 3(1). For
example, recognizing sequences of an appropriate restriction enzyme
are introduced into the 5'-terminals of both terminals of a
synthetic DNA fragment, among the synthetic DNA fragments which are
used in the item 3(5) for constructing the VH and VL of the human
CDR-grafted antibody, so that they are cloned into upstream of the
genes encoding CH and CL of a human antibody in the vector for
expression of humanized antibody described in the item 3(1) in such
a manner that they can be expressed in a suitable form, to thereby
construct the human CDR-grafted antibody expression vector.
[0515] (7) Stable Production of Humanized Antibody
[0516] A transformant capable of stably producing a human chimeric
antibody and a human CDR-grafted antibody (both hereinafter
referred to as "humanized antibody") can be obtained by introducing
the vectors for humanized antibody expression described in the
items 3(4) and (6) into an appropriate animal cell.
[0517] The method for introducing a humanized antibody expression
vector into an animal cell includes electroporation [Japanese
Published Unexamined Patent Application No. 257891/90,
Cytotechnology, 3, 133 (1990)] and the like.
[0518] As the animal cell into which a humanized antibody
expression vector is introduced, any cell can be used so long as it
is an animal cell which can produce the humanized antibody.
[0519] Examples include mouse myeloma cells such as NS0 cell and
SP2/0 cell, Chinese hamster ovary cells such as CHO/dhfr.sup.- cell
and CHO/DG44 cell, rat myeloma such as YB2/0 cell and IR983F cell,
BHK cell derived from a syrian hamster kidney, a human myeloma cell
such as Namalwa cell, and the like, and a Chinese hamster ovary
cell CHO/DG44 cell, a rat myeloma YB2/0 cell and the host cells of
the present invention described in the item 5 are preferred.
[0520] After introduction of the humanized antibody expression
vector, a transformant capable of stably producing the humanized
antibody can be selected by using a medium for animal cell culture
comprising an agent such as G418 sulfate (hereinafter referred to
as "G418"; manufactured by SIGMA) and the like in accordance with
the method described in Japanese Published Unexamined Patent
Application No. 257891/90. The medium to culture animal cells
includes RPMI 1640 medium (manufactured by Nissui Pharmaceutical),
GIT medium (manufactured by Nihon Pharmaceutical), EX-CELL 302
medium (manufactured by JRH), IMDM medium (manufactured by GIBCO
BRL), Hybridoma-SFM medium (manufactured by GIBCO BRL), media
obtained by adding various additives such as fetal bovine serum
(hereinafter referred to as "FBS") to these media, and the like.
The humanized antibody can be produced and accumulated in the
culture supernatant by culturing the obtained transformant in a
medium. The amount of production and antigen binding activity of
the humanized antibody in the culture supernatant can be measured
by a method such as enzyme-linked immunosorbent assay (hereinafter
referred to as "ELISA"; Antibodies, Monoclonal Antibodies) or the
like. Also, the amount of the humanized antibody produced by the
transformant can be increased by using a DHFR gene amplification
system in accordance with the method described in Japanese
Published Unexamined Patent Application No. 257891/90.
[0521] The humanized antibody can be purified from a culture
supernatant culturing the transformant by using a protein A column
(Antibodies, Chapter 8; Monoclonal Antibodies). In addition,
purification methods generally used for the purification of
proteins can also be used. For example, the purification can be
carried out through the combination of gel filtration, ion exchange
chromatography and ultrafiltration. The molecular weight of the H
chain, L chain or antibody molecule as a whole of the purified
humanized antibody can be measured, e.g., by polyacrylamide gel
electrophoresis [hereinafter referred to as "SDS-PAGE", Nature,
227, 680 (1970)], Western blotting (Antibodies, Monoclonal
Antibodies) or the like.
[0522] B. Preparation of Fc Fusion Protein
[0523] (1) Construction of Fc Fusion Protein Expression Vector
[0524] An Fc fusion protein expression vector is an expression
vector for animal cells into which genes encoding the Fc region of
a human antibody and a protein to be fused are inserted, which can
be constructed by cloning each of genes encoding the Fc region of a
human antibody and the protein to be fused into an expression
vector for animal cell.
[0525] The Fc region of a human antibody includes those containing
a part of a hinge region and/or CH1 in addition to regions
containing CH2 and CH3 regions. Also, it can be any Fc region, so
long as at least one amino acid of CH2 or CH3 may be deleted,
substituted, added or inserted, and substantially has the binding
activity to the Fc.gamma. receptor.
[0526] As the genes encoding the Fc region of a human antibody and
the protein to be fused, a chromosomal DNA comprising an exon and
an intron can be used, and a cDNA can also be used. The method for
linking the genes and the Fc region includes PCR using each of the
gene sequences as the template (Molecular Cloning, Second Edition,
Current Protocols in Molecular Biology, Supplement 1-34).
[0527] As the expression vector for animal cell, any vector can be
used, so long as a gene encoding the C region of a human antibody
can be inserted thereinto and expressed therein. Examples include
pAGE107 [Cytotechnology, 3, 133 (1990)], pAGE103 [J. Biochem., 101,
1307 (1987)], pHSG274 [Gene, 27, 223 (1984)], pKCR [Proc. Natl.
Acad. Sci. USA 78, 1527 (1981), pSG1 .beta. d2-4 [Cytotechnology,
4, 173 (1990)] and the like. The promoter and enhancer in the
expression vector for animal cell include SV40 early promoter and
enhancer a [J. Biochem., 101, 1307 (1987)], Moloney mouse leukemia
virus LTR [Biochem. Biophys. Res. Commun., 149, 960 (1987)],
immunoglobulin H chain promoter [Cell, 41, 479 (1985)] and enhancer
[Cell, 33, 717 (1983)], and the like.
[0528] (2) Preparation of DNA Encoding Fc Region of Human Antibody
and Protein to be Fused
[0529] A DNA encoding the Fc region of a human antibody and the
protein to be fused can be obtained in the following manner.
[0530] A cDNA is synthesized from mRNA extracted from a cell or
tissue which expresses the protein of interest to be fused with Fc.
The synthesized cDNA is cloned into a vector such as a phage or a
plasmid to obtain a cDNA library. A recombinant or recombinant
plasmid comprising cDNA encoding the protein of interest is isolate
from the library using the gene sequence part of the protein of
interest as the probe. A full nucleotide sequence of the antibody
of interest on the recombinant phage or recombinant plasmid is
determined, and a full length amino acid sequence is deduced from
the nucleotide sequence.
[0531] As the non-human animal, any animal such as mouse, rat,
hamster or rabbit can be used, so long as a cell or tissue can be
removed therefrom.
[0532] The method for preparing a total RNA from a cell or tissue
includes the guanidine thiocyanate-cesium trifluoroacetate method
[Method in Enzymology, 154, 3 (1987)] and the like, and the method
for preparing mRNA from total A includes an oligo (dT)-immobilized
cellulose column method (Molecular Cloning, Second Edition) and the
like. In addition, a kit for preparing mRNA from a cell or tissue
includes Fast Track mRNA Isolation Kit (manufactured by
Invitrogen), Quick Prep mRNA Purification Kit (manufactured by
Pharmacia) and the like.
[0533] The method for synthesizing a cDNA and preparing a cDNA
library includes the usual methods (Molecular Cloning, Second
Edition; Current Protocols in Molecular Biology, Supplement 1-34);
methods using a commercially available kit such as SuperScript.TM.,
Plasmid System for cDNA Synthesis and Plasmid Cloning (manufactured
by GIBCO BRL) or ZAP-cDNA Synthesis Kit (manufactured by
Stratagene); and the like.
[0534] In preparing the cDNA library, the vector into which a cDNA
synthesized using mRNA extracted from a cell or tissue as the
template is inserted may be any vector so long as the DNA can be
inserted. Examples include ZAP Express [Strategies, 5, 58 (1992)],
pBluescript II SK(+) [Nucleic Acids Research, 17, 9494 (1989)],
.lambda.zapII (manufactured by Stratagene), .lambda.gt10 and
.lambda.gt11 [DNA Cloning, A Practical Approach, I, 49 (1985)],
Lambda BlueMid (manufactured by Clontech), .lambda.ExCell, pT7T318U
(manufactured by Pharmacia), pcD2 [Mol. Cell. Biol., 3, 280
(1983)], pUC18 [Gene, 33, 103 (1985)] and the like.
[0535] As Escherichia coli into which the cDNA library constructed
from a phage or plasmid vector is introduced, any Escherichia coli
can be used, so long as the cDNA library can be introduced,
expressed and maintained. Examples include XL1-Blue MRF'
[Strategies, 5, 81 (1992)], C600 [Genetics, 39, 440 (1954)], Y1088
and Y1090 [Science, 222, 778 (1983)], NM522 [J. Mol. Biol., 166, 1
(1983)], K802 [J. Mol. Biol. 16, 118 (1966)], JM105 [Gene, 38, 275
(1985)] and the like.
[0536] As the method for selecting a cDNA clone encoding the
protein of interest from the cDNA library, a colony hybridization
or a plaque hybridization using an isotope- or fluorescence-labeled
probe can be used (Molecular Cloning, Second Edition). The cDNA
encoding the protein of interest can also be prepared by preparing
primers and using a cDNA synthesized from mRNA or a cDNA library as
the template according to PCR.
[0537] The method for fusing the protein of interest with the Fc
region of a human antibody includes PCR. For example, synthesized
oligo DNAs (primers) are designed at the 5'-terminal and
3'-terminal of the gene sequence encoding the protein of interest,
and PCR is carried out to prepare a PCR product. In the same
manner, primers are designed for the gene sequence encoding the Fc
region of a human antibody to be fused to prepare a PCR product. At
this time, the primers are designed in such a manner that the same
restriction enzyme site or the same gene sequence is present
between the 3'-terminal of the PCR product of the protein to be
fused and the 5'-terminal of the PCR product of the Fc region. When
it is necessary to modify the amino acids around the linked site,
mutation is introduced by using the primer into which the mutation
is introduced. PCR is further carried out by using the two kinds of
the obtained PCR fragments to link the genes. Also, they can be
linked by carrying out ligation after treatment with the same
restriction enzyme.
[0538] The nucleotide sequence of the DNA can be determined by
digesting the gene sequence linked by the above method with
appropriate restriction enzymes, cloning the fragments into a
plasmid such as pBluescript SK(-) (manufactured by Stratagene),
carrying out analysis by using a generally used nucleotide sequence
analyzing method such as the dideoxy method of Sanger et al. [Proc.
Natl. Acad. Sci. USA, 74, 5463 (1977)] or an automatic nucleotide
sequence analyzer such as ABI PRISM 377 DNA Sequencer (manufactured
by Pharmacia).
[0539] Whether or not the obtained cDNA encodes the full length
amino acid sequences of the Fc fusion protein containing a
secretory signal sequence can be confirmed by deducing the full
length amino acid sequence of the Fc fusion protein from the
determined nucleotide sequence and comparing it with the amino acid
sequence of interest.
[0540] (3) Stable Production of Fc Fusion Protein
[0541] A transformant capable of stably producing an Fc fusion
protein can be obtained by introducing the Fc fusion protein
expression vector described in the item (1) into an appropriate
animal cell.
[0542] The method for introducing the Fc fusion protein expression
vector into an animal cell include electroporation [Japanese
Published Unexamined Patent Application No. 257891/90,
Cytotechnology, 3, 133 (1990)] and the like.
[0543] As the animal cell into which the Fc fusion protein
expression vector is introduced, any ell can be used, so long as it
is an animal cell which can produce the Fc fusion protein.
[0544] Examples include mouse myeloma cells such as NS0 cell and
SP2/0 cell, Chinese hamster ovary cells such as CHO/dhfr.sup.- cell
and CHO/D44 cell, rat myeloma such as YB2/0 cell and IR983F cell,
BHK cell derived from a syrian hamster kidney, a human myeloma cell
such as Namalwa cell, and the like, and preferred are a Chinese
hamster ovary cell CHO/DG44 cell, a rat myeloma YB2/0 cell and the
host cells used in the method of the present invention described in
the item 1.
[0545] After introduction of the Fc fusion protein expression
vector, a transformant capable of stably producing the Fc fusion
protein expression vector can be selected by using a medium for
animal cell culture comprising an agent such as G418 and the like
in accordance with the method described in Japanese Published
Unexamined Patent Application No. 257891/90. The medium to culture
animal cells includes RPMI 1640 medium (manufactured by Nissui
Pharmaceutical), GIT medium (manufactured by Nihon Pharmaceutical),
EX-CELL 302 medium (manufactured by JRH), IMDM medium (manufactured
by GIBCO BRL), Hybridoma-SFM medium (manufactured by GIBCO BRL),
media obtained by adding various additives such as fetal bovine
serum to these media, and the like. The Fc fusion protein can be
produced and accumulated in the culture medium by culturing the
obtained transformant in a medium. The production amount and
antigen binding activity of the Fc fusion protein in the culture
medium can be measured by a method such as ELISA. Also, the amount
of the Fc fusion protein produced by the transformant can be
increased by using a dhfr gene amplification system in accordance
with the method described in Japanese Published Unexamined Patent
Application No. 257891/90.
[0546] The Fc fusion protein can be purified from a culture
supernatant culturing the transformant using a protein A column
(Antibodies, Chapter 8, Monoclonal Antibodies). In addition,
purification methods generally used for purifying proteins can also
be used. For example, the purification can be carried out through
the combination of a gel filtration, an ion exchange chromatography
and an ultrafiltration. The molecular weight as a whole of the
purified Fc fusion protein molecule can be measured by SDS-PAGE
[Nature, 227, 680 (1970)], Western blotting (Antibodies, Chapter
12; Monoclonal Antibodies) or the like.
[0547] Thus, methods for producing an antibody composition using an
animal cell as the host cell have been described, but, as described
above, it can also be produced by yeast, an insect cell, a plant
cell, an animal individual or a plant individual by the same
methods on the animal cell.
[0548] When the host cell is capable of preparing the antibody
molecule, the antibody composition of the present invention can be
prepared by culturing the cell capable of expressing an antibody
molecule according to the method described in the above item 1,
culturing the cell, and recovering the antibody composition of
interest.
[0549] 4. Measurement of Binding Activity to Human
Fc.gamma.RIIIa
[0550] Binding activity of the antibody composition to
Fc.gamma.RIIIa can be measured by the following technique.
[0551] (1) Preparation of Human Fc.gamma.RIIIa
[0552] Fc.gamma.RIIIa which can be used includes Fc.gamma.IIIa
present on the cell surface of peripheral blood lymphocyte of a
human or non-human animal, Fc.gamma.IIIa obtained by preparing a
gene encoding Fc.gamma.RIIIa and introducing the gene into a host
cell and expressing the Fc.gamma.R on the cell surface,
Fc.gamma.RIIIa secreted from the cell, and the like.
[0553] A method for preparing a gene encoding Fc.gamma.RIIIa,
introducing the gene into a host cell and expressing the
Fc.gamma.RIIIa on the cell surface, and a method obtaining
Fc.gamma.RIIIa by secreting it from the cell are described
below.
[0554] A total RNA or mRNA is prepared from human or non-human
animal tissues or cells.
[0555] A commercially available product (e.g., manufactured by
Clontech) can be used as the mRNA of human or non-human animal
tissues or cells, or it may be prepared from human or non-human
animal tissues or cells as follows. The method for preparing a
total RNA from human or non-human animal tissues or cells includes
the guanidine thiocyanate-cesium trifluoroacetate method [Methods
in Enzymology, 154, 3 (1987),], the acidic guanidine thiocyanate
phenol chloroform (AGPC) method [Analytical Biochemistry, 162, 156
(1987); Experimental Medicine (Jikken Igaku), 9, 1937 (1991)] and
the like.
[0556] Also, the method for preparing mRNA as poly(A).sup.+ RNA
from a total RNA includes an oligo(dT)-immobilized cellulose column
method (Molecular Cloning, Second Edition) and the like.
[0557] In addition, mRNA can be prepared by using a kit such as
Fast Track mRNA Isolation Kit (manufactured by Invitrogen) or Quick
Prep mRNA Purification Kit (manufactured by Pharmacia).
[0558] A cDNA library is prepared from a full mRNA or mRNA of the
prepared human or non-human animal tissue or cell.
[0559] The method for preparing a cDNA library include methods
described in Molecular Cloning, Second Edition, Current Protocols
in Molecular Biology, and the like, methods using a commercially
available kit such as SuperScript Plasmid System for cDNA Synthesis
and Plasmid Cloning (manufactured by Life Technologies) or ZAP-cDNA
Synthesis Kit (manufactured by STRATAGENE), and the like.
[0560] As the cloning vector for the preparation of the cDNA
library, any vector such as a phage vector or a plasmid vector can
be used, so long as it is autonomously replicable in Escherichia
coli K12. Examples include ZAP Express (manufactured by STRATAGENE,
Strategies, 5, 58 (1992)), pBluescript II SK(+) [Nucleic Acids
Research, 17, 9494 (1989)], Lambda ZAP II (manufactured by
STRATAGENE), .lambda.gt10 and .lambda.gt11 [DNA Cloning, A
Practical Approach, 1, 49 (1985)], .lambda.TriplEx (manufactured by
Clontech), .lambda.ExCell (manufactured by Pharmacia), pcD2 [Mol.
Cell. Biol., 3, 280 (1983)], pUC18 [Gene, 33, 103 (1985)] and the
like.
[0561] Any microorganism can be used as the host microorganism, and
Escherichia coli is preferably used. Examples include Escherichia
coli XL1-Blue MRF' [manufactured by STRATAGENE, Strategies, 5, 81
(1992)], Escherichia coli C600 [Genetics, 39, 440 (1954)],
Escherichia coli Y1088 [Science, 222, 778 (1983)], Escherichia coli
Y1090 [Science, 222, 778 (1983)], Escherichia coli NM522 [J. Mol.
Biol. 166, 1 (1983)], Escherichia coli K802 [J. Mol. Biol, 16, 118
(1966)], Escherichia coli JM105 [Gene, 38, 275 (1985)] and the
like.
[0562] The cDNA library may be used as such in the succeeding
analysis, and in order to obtain a full length cDNA as efficient as
possible by decreasing the ratio of an infull length cDNA, a cDNA
library prepared using the oligo cap method developed by Sugano et
al. [Gene, 138, 171 (1994); Gene, 200, 149 (1997), Protein, Nucleic
Acid and Enzyme, 41, 603 (1996); Experimental Medicine, 11, 2491
(1993); cDNA Cloning (Yodo-sha) (1996); Methods for Preparing Gene
Libraries (Yodo-sha) (1994)] may be used in the following
analysis.
[0563] A gene encoding Fc.gamma.R can be obtained by preparing
primers specific for 5'-terminal and 3'-terminal nucleotide
sequences based on the nucleotide sequences of various
Fc.gamma.RIIIa, and amplifying DNA by PCR [PCR Protocols, Academic
Press (1990)] using a prepared cDNA library as the template.
[0564] Whether the thus obtained gene is a DNA encoding
Fc.gamma.RIIIa can be confirmed by analyzing it according to the
generally used nucleotide sequence analyzing method such as the
dideoxy method of Sanger et al. [Proc. Natl. Acad. Sci. U.S.A., 74,
5463 (1977)] or by using a nucleotide sequence analyzer such as ABI
PRISM 377 DNA Sequencer (manufactured by PE Biosystems).
[0565] The nucleotide sequence of a gene encoding Fc.gamma.RIIIa
obtained by the above method includes the nucleotide sequence of
Fc.gamma.RIIIa represented by SEQ ID NO:27.
[0566] The gene encoding Fc.gamma.RIIIa can also be obtained based
on the determined DNA nucleotide sequence by carrying out chemical
synthesis by a DNA synthesizer such as DNA Synthesizer Model 392
manufactured by Perkin Elmer using a phosphoamidite method.
[0567] A recombinant vector is prepared by inserting the thus
obtained cDNA encoding Fc.gamma.RIIa into downstream of the
promoter of an appropriate expression vector.
[0568] A transformant which produces an antibody molecule can be
obtained by introducing the recombinant vector into a host cell
suitable for the expression vector.
[0569] As the host cell, any of yeast, an animal cell, an insect
cell, a plant cell or the like can be used, so long as it can
express the gene of interest.
[0570] As the expression vector, a vector which is autonomously
replicable in the above host cell or can be integrated into the
chromosome and comprises a promoter at such a position that the DNA
encoding the Fc.gamma.RIIIa of interest can be transferred is
used.
[0571] When a yeast is used as the host cell, the expression vector
includes YEP13 (ATCC 37115), YEp24 (ATCC 37051), YCp50 (ATCC 37419)
and the like.
[0572] Any promoter can be used, so long as it can function in
yeast. Examples include a promoter of a gene of the glycolytic
pathway such as a hexose kinase gene, PHOS promoter, PGK promoter,
GAP promoter, ADH promoter, gal 1 promoter, gal 10 promoter, heat
shock protein promoter, MF .alpha.1 promoter, CUP 1 promoter and
the like.
[0573] The host cell includes microorganisms belonging to the genus
Saccharomyces, the genus Schizosaccharomyces, the genus
Kluyveromyces, the genus Trichosporon, the genus Schwanniomyces and
the like, such as Saccharomyces cerevisiae, Schizosaccharomyces
pombe, Kluyveromyces lactis, Trichosporon pullulans and
Schwanniomyces alluvius.
[0574] As the method for introducing the recombinant vector, any
method can be used, so long as it can introduce DNA into yeast.
Examples include electroporation [Method in Enzymology, 194, 182
(1990)], spheroplast method [Proc. Natl. Acad. Sci. USA, 84, 1929
(1978)], lithium acetate method [J. Bacteriol., 153, 163 (1983)), a
method described in Proc. Natl. Acad. Sci. USA, 75, 1929 (1978) and
the like.
[0575] When an animal cell is used as the host, the expression
vector includes pcDNAI, pcDM8 (available from Funakoshi), pAGE107
Japanese Published Unexamined Patent Application No. 22979/91;
Cytotechnology, 3, 133 (1990)], pAS3-3, (Japanese Published
Unexamined Patent Application No. 227075/90), pCDM8 [Nature,
329-840 (1987)], pcDNAI/Amp (manufactured by Invitrogen), pREP4
(manufactured by Invitrogen), pAGE103 [J. Biochemistry, 101, 1307
(1987)], pAGE210 and the like.
[0576] Any promoter can be used, so long as it can function in an
animal cell. Examples include a promoter of IE (immediate early)
gene of cytomegalovirus (CMV), an early promoter of SV40, a
promoter of retrovirus, a promoter of metallothionein, a heat shock
promoter, an SR.alpha. promoter and the like. Also, an enhancer of
the IE gene of human CMV may be used together with the
promoter.
[0577] The host cell includes a human cell such as Namalwa cell, a
monkey cell such as COS cell, a Chinese hamster cell such as CHO
cell or HBT5637 (Japanese Published Unexamined Patent Application
No. 299/88), a rat myeloma cell, a mouse myeloma cell, a cell
derived from syrian hamster kidney, an embryonic stem cell, a
fertilized egg cell and the like.
[0578] As the method for introducing the recombinant vector, any
method can be used, so long as it can introduce DNA into an animal
cell. Examples include electroporation [Cytotechnology, 3, 133
(1990)], the calcium phosphate method (Japanese Published
Unexamined Patent Application No. 227075/90), the lipofection
method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)], the injection
method (Manipulating the Mouse Embryo, A Laboratory Manual), a
method using particle gun (gene gun) (Japanese Patent No. 2606856,
Japanese Patent No. 2517813), the DEAE-dextran method [Biomanual
Series 4--Gene Transfer and Expression Analysis (Yodo-sha), edited
by Takashi Yokota and Kenichi Arai (1994)], the virus vector method
(Manipulating Mouse Embryo, Second Edition) and the like.
[0579] When an insect cell is used as the host, the protein can be
expressed by the method described in Current Protocols in Molecular
Biology, Baculovirus Expression Vectors, A Laboratory Manual, W.H.
Freeman and Company, New York (1992), Bio/Technology, 6, 47 (1988)
or the like.
[0580] That is, the protein can be expressed by co-introducing a
recombinant gene-introducing vector and a baculovirus into an
insect cell to obtain a recombinant virus in an insect cell culture
supernatant and then infecting the insect cell with the recombinant
virus.
[0581] The gene introducing vector used in the method includes
pVL1392, pVL1393, pBlueBacIII (all manufactured by Invitrogen) and
the like.
[0582] The baculovirus includes Autographa californica nuclear
polyhedrosis virus which is infected by an insect of the family
Barathra.
[0583] The insect cell includes Spodoptera frugiperda oocytes Sf9
and Sf1 [Current Protocols in Molecular Biology, Baculovirus
Expression Vectors, A Laboratory Manual, W.H. Freeman and Company,
New York (1992)], a Trichoplusia ni oocyte High 5 (manufactured by
Invitrogen) and the like.
[0584] The method for co-introducing the recombinant
gene-introducing vector and the baculovirus for preparing the
recombinant virus includes the calcium phosphate method (Japanese
Published Unexamined Patent Application No. 227075/90), the
lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)]
and the like.
[0585] When a plant cell is used as the host, the expression vector
includes Ti plasmid, tobacco mosaic virus and the like.
[0586] As the promoter, any promoter can be used, so long as it can
function in a plant cell. Examples include cauliflower mosaic virus
(CaMV) 35S promoter, rice actin 1 promoter and the like.
[0587] The host cell includes plant cells of tobacco, potato,
tomato, carrot, soybean, rape, alfalfa, rice, wheat, barley and the
like.
[0588] As the method for introducing the recombinant vector, any
method can be used, so long as it can introduce DNA into a plant
cell. Examples include a method using Agrobacterium (Japanese
Published Unexamined Patent Application No. 140885/84, Japanese
Published Unexamined Patent Application No. 70080/85, WO94/00977),
electroporation (Japanese Published Unexamined Patent Application
No. 251887/85), a method using a particle gun (gene gun) (Japanese
Patent No. 2606856, Japanese Patent No. 2517813) and the like.
[0589] As the method for expressing a gene, secretion production,
expression of a fusion protein with other protein and the like can
be carried out in accordance with the method described in Molecular
Cloning, Second Edition or the like, in addition to the direct
expression.
[0590] Fc.gamma.RIIIa can be produced by culturing the thus
obtained transformant in a medium to produce and accumulate
Fc.gamma.RIIIa in the culture and then recovering it from the
resulting culture. The method for culturing the transformant using
a medium can be carried out in accordance with a general method
which is used for culturing host cells.
[0591] As the medium for culturing a transformant obtained by using
yeast as the host, the medium may be either a natural medium or a
synthetic medium, so long as efficiently carried out.
[0592] As the carbon source, those which can be assimilated by the
yeast can be used. Examples include carbohydrates such as glucose,
fructose, sucrose, molasses containing them, starch and starch
hydrolysate; organic acids such as acetic acid and propionic acid;
alcohols such as ethanol and propanol; and the like.
[0593] The nitrogen source includes ammonia; ammonium salts of
inorganic acid or organic acid such as ammonium chloride, ammonium
sulfate, ammonium acetate and ammonium phosphate, other
nitrogen-containing compounds; peptone; meat extract; yeast
extract; corn steep liquor; casein hydrolysate; soybean meal;
soybean meal hydrolysate; various fermented cells and hydrolysates
thereof; and the like.
[0594] The inorganic salt includes potassium dihydrogen phosphate,
dipotassium hydrogen phosphate, magnesium phosphate, magnesium
sulfate, sodium chloride, ferrous sulfate, manganese sulfate,
copper sulfate, calcium carbonate, and the like.
[0595] The culturing is carried out generally under aerobic
conditions such as shaking culture or submerged-aeration stiffing
culture. The culturing temperature is preferably 15 to 40.degree.
C., and the culturing time is generally 16 hours to 7 days. During
the culturing, the pH is maintained at 3.0 to 9.0. The pH is
adjusted with an inorganic or organic acid, an alkali solution,
urea, calcium carbonate, ammonia or the like.
[0596] If necessary, an antibiotic such as ampicillin or
tetracycline can be added to the medium during the culturing.
[0597] When yeast transformed with a recombinant vector obtained by
using an inducible promoter as the promoter is cultured, an inducer
can be added to the medium, if necessary. For example, when yeast
transformed with a recombinant vector obtained using lac promoter
is cultured, isopropyl-.beta.-D-thiogalactopyranoside can be added
to the medium, and when yeast transformed with a recombinant vector
obtained using trp promoter is cultured, indoleacrylic acid and the
like can be added to the medium.
[0598] When a transformant obtained by using an animal cell as the
host cell is cultured, the medium includes generally used RPMI 1640
medium [The Journal of the American Medical Association, 199, 519
(1967)], Eagle's MEM medium [Science, 122, 501 (1952)], Dulbecco's
modified MEM medium [Virology, 8, 396 (1959)], 199 medium
[Proceeding of the Society for the Biological Medicine, 73, 1
(1950)] and Whitten's medium [Developmental Engineering
Experimentation Manual (Hassei Kogaku Jikken Manual)--Preparation
of Transgenic Mice (Kodan-sha), edited by M. Katsuki (1987)], the
media to which fetal calf serum, etc. is added, and the like.
[0599] The culturing is carried out generally at a pH of 6.0 to 8.0
and 30 to 40.degree. C. for 1 to 7 days in the presence of 5%
CO.sub.2. If necessary, an antibiotic such as kanamycin or
penicillin can be added to the medium during the culturing.
[0600] The medium for the culturing of a transformant obtained by
using an insect cell as the host includes generally used TNM-FH
medium (manufactured by Pharmingen), Sf-900 II SFM medium
(manufactured by Life Technologies), ExCell 400 and ExCell 405
(both manufactured by JRH Biosciences), Grace's Insect Medium
[Nature 195, 788 (1962)] and the like.
[0601] The culturing is carried out generally at a medium pH of 6.0
to 7.0 and 25 to 30.degree. C. for 1 to 5 days.
[0602] In addition antibiotics such as gentamicin can be added to
the medium during the culturing, if necessary.
[0603] A transformant obtained by using a plant cell as the host
cell can be cultured as a cell or by differentiating it into a
plant cell or organ. The medium for culturing the transformant
includes generally used Murashige and Skoog (MS) medium and White
medium, the media to which a plant hormone such as auxin or
cytokinin is added, and the like.
[0604] The culturing is carried out generally at a pH of 5.0 to 9.0
and 20 to 40.degree. C. for 3 to 60 days.
[0605] If necessary, an antibiotic such as kanamycin or hygromycin
can be added to the medium during the culturing.
[0606] As discussed above, Fc.gamma.RIIIa can be produced by
culturing a transformant derived from a microorganism, an animal
cell, an insect cell or a plant cell which comprises a recombinant
vector into which a DNA encoding Fc.gamma.RIIIa is inserted, in
accordance with the general culturing method to thereby produce and
accumulate Fc.gamma.RIIIa, and then recovering Fc.gamma.RIIIa from
the culture.
[0607] As the method for expressing Fc.gamma.RIIIa, secretion
production, expression of fusion protein and the like can be
carried out in accordance with the method described in Molecular
Cloning, Second Edition in addition to the direct expression.
[0608] The method for producing Fc.gamma.RIIIa includes a method of
intracellular expression in a host cell, a method of extracellular
secretion from a host cell, and a method of production on a host
cell membrane outer envelope. The method can be selected by
changing the host cell used or the structure of Fc.gamma.RIIIa
produced.
[0609] When Fc.gamma.RIIa is produced in a host cell or on a host
cell membrane outer envelope, it can be positively secreted
extracellularly in accordance with the method of Paulson et al. [J.
Biol. Chem., 264, 17619 (1989)], the method of Lowe et al. [Proc.
Natl. Acad. Sci. USA, 86, 8227 (1989), Genes Develop., 4, 1288
(1990)], the methods described in Japanese Published Unexamined
Patent Application No. 336963/93 and Japanese Published Unexamined
Patent Application No. 823021/94 and the like.
[0610] That is, Fc.gamma.RIIIa of interest can be positively
secreted extracellularly from a host cell by inserting a DNA
encoding Fc.gamma.RIIIa and a signal peptide suitable for the
expression of Fc.gamma.RIIIa into an expression vector by using a
gene recombination technique, and then expressing the vector.
[0611] Also, its production amount can be increased in accordance
with the method described in Japanese Published Unexamined Patent
Application No. 227075/90 based on a gene amplification system
using a dihydrofolate reductase gene.
[0612] In addition, Fc.gamma.R can also be produced by using a
gene-introduced animal individual (transgenic non-human animal) or
a plant individual (transgenic plant) which is constructed by the
redifferentiation of an animal or plant cell into which the gene is
introduced.
[0613] When the transformant is an animal individual or a plant
individual, Fc.gamma.RIIIa can be produced in accordance with a
general method by rearing or cultivating it to thereby produce and
accumulate Fc.gamma.R and then recovering Fc.gamma.RIIa from the
animal or plant individual.
[0614] The method for producing Fc.gamma.RIIIa by using an animal
individual includes a method in which Fc.gamma.RIIIa of interest is
produced in an animal constructed by introducing a gene in
accordance with a known method [American Journal of Clinical
Nutrition, 63, 639 (1996); American Journal of Clinical Nutrition,
63, 627S (1996); Bio/Technology, 9, 830 (1991)].
[0615] In the case of an animal individual, Fc.gamma.RIIIa can be
produced by rearing a transgenic non-human animal into which a DNA
encoding Fc.gamma.RIIIa is introduced to thereby produce and
accumulate Fc.gamma.RIIIa in the animal, and then recovering
Fc.gamma.RIIIa from the animal. The place of the animal where
Fc.gamma.RIIIa is produced and accumulated includes milk (Japanese
Published Unexamined Patent Application No. 309192/88) and eggs of
the animal. As the promoter used in this case, any promoter can be
used, so long as it can function in an animal. Preferred examples
include mammary gland cell-specific promoters such as .beta. casein
promoter, casein promoter, .beta. lactoglobulin promoter and whey
acidic protein promoter.
[0616] The method for producing Fc.gamma.RIIIa by using a plant
individual includes a method in which Fc.gamma.RIIIa is produced by
cultivating a transgenic plant into which a DNA encoding Fc.gamma.R
is introduced by a known method [Tissue Culture (Soshiki Baiyo), 20
(1994); Tissue Culture (Soshiki Baiyo), 21 (1995); Trends in
Biotechnology, 15, 45 (1997)] to produce and accumulate Fc.gamma.R
in the plant, and then recovering Fc.gamma.RIIIa from the
plant.
[0617] Regarding purification of Fc.gamma.RIIIa produced by a
transformant into which a gene encoding Fc.gamma.RIIa is
introduced, for example, when Fc.gamma.RIIIa is intracellularly
expressed in a dissolved state, the cells after culturing are
recovered by centrifugation, suspended in an aqueous buffer and
then disrupted using ultrasonic oscillator, French press, Manton
Gaulin homogenizer, dynomill or the like to obtain a cell-free
extract. A purified product of Fc.gamma.RIIa can be obtained from a
supernatant obtained by centrifuging the cell-free extract, by
using an ordinary enzyme isolation purification technique such as
solvent extraction; salting out and desalting with ammonium
sulfate, etc. precipitation with an organic solvent; anion exchange
chromatography using a resin such as diethylaminoethyl
(DEAE)-Sepharose or DIAION HPA-75 (manufactured by Mitsubishi
Chemical); cation exchange chromatography using a resin such as
S-Sepharose FF (manufactured by Pharmacia); hydrophobic
chromatography using a resin such as butyl-Sepharose or
phenyl-Sepharose; gel filtration using a molecular sieve, affinity
chromatography; chromatofocusing; electrophoresis such as
isoelectric focusing; and the like which may be used alone or in
combination.
[0618] Also, when Fc.gamma.RIIIa is expressed intracellularly by
forming an insoluble body, the cells are recovered, disrupted and
centrifuged in the same manner, and the insoluble body of
Fc.gamma.RIIa is recovered as a precipitation fraction. The
recovered insoluble body of Fc.gamma.RIIIa is solubilized by using
a protein denaturing agent. Fc.gamma.RIIIa is made into a normal
three-dimensional structure by diluting or dialyzing the
solubilized solution, and then a purified product of Fc.gamma.RIIIa
is obtained by the same isolation purification method.
[0619] When Fc.gamma.RIIa is secreted extracellularly,
Fc.gamma.RIIa or derivatives thereof can be recovered from the
culture supernatant. That is, the culture is treated by a similar
technique such as centrifugation to obtain a soluble fraction, and
a purified preparation of Fc.gamma.RIIa can be obtained from the
soluble fraction by the same isolation purification method.
[0620] (2) Measurement of Binding Activity to Fc.gamma.RIIIa
[0621] Binding activity of the antibody composition to
Fc.gamma.RIIIa expressed on the cell membrane can be measured by
the immunofluorescent method [Cancer Immunol. Immunother., 36, 373
(1993)] or the like. Also, binding activity to the purified
Fc.gamma.RIIa prepared by the method described in the item 4(1) can
be measured according to the immunological determination method
such as Western staining described in literatures [Monoclonal
Antibodies: Principles and Applications, Wiley-Liss, Inc., (1995);
Enzyme Immunoassay (Koso Men-eki Sokutei Ho), 3rd edition, Igaku
Shoin (1987), reversed edition; Enzyme Antibody Method (Koso Kotai
Ho), Gakusai Kikaku (1985)], RIA (radioimmunoassay), VIA
(viroimmunoassay), EIA (enzyme or immunoassay), FIA
(fluoroimmunoassay) or MIA (metalloimmunoassay), for example, as
follows.
[0622] Fc.gamma.RIIIa is immobilized on a plastic plate for EIA and
is allowed to react with a sample containing an antibody
composition. Next, an amount of the bound antibody composition is
measured by using an appropriate secondary antibody.
[0623] In addition, binding activity to the purified Fc.gamma.IIIa
can also be measured by a measuring method using biosensor [e.g.,
BIAcore (manufactured by BIACORE)] [J. Immunol. Methods, 200, 121
(1997)], isothermal titration calorimetry [Proc. Natl. Acad. Sci.
U.S.A., 97, 9026 (2000)] or the like.
[0624] 5. Activity Evaluation of Antibody Composition
[0625] As the method for measuring the amount of the purified
antibody composition, its binding activity to an antigen, its
binding activity to Fc.gamma.RIIIa and its effector function, the
known method described in Monoclonal Antibodies, Antibody
Engineering and the like can be used.
[0626] For example, when the antibody composition is a humanized
antibody, the binding activity to an antigen, binding activity to
an antigen-positive cultured clone and binding activity to
Fc.gamma.RIIIa can be measured by methods such as ELISA and the
immunofluorescent method [Cancer Immunol, Immunother., 36, 373
(1993)], measurement using biosensor [for example, using BIAcore
(manufactured by BIACORE)] [J. Immunol. Methods, 200, 121 (1997)],
isothermal titration calorimetry method [Proc. Natl. Acad. Sci.
U.S.A., 97, 9026 (2000)] and the like. Among the effector
functions, the cytotoxic activity against an antigen-positive
cultured clone can be evaluated by measuring CDC activity, ADCC
activity [Cancer Immunol. Immunother., 36, 373 (1993)] and the
like.
[0627] 6. Analysis of Sugar Chains of Antibody Molecule Expressed
in Various Cells
[0628] The sugar chain structure binding to an antibody molecule
expressed in various cells can be analyzed in accordance with the
general analysis of the sugar chain structure of a glycoprotein.
For example, the sugar chain which is bound to IgG molecule
comprises a neutral sugar such as galactose, mannose, fucose, an
amino sugar such as N-acetylglucosamine and an acidic sugar such as
sialic acid, and can be analyzed by a method such as a sugar chain
structure analysis using sugar composition analysis, two
dimensional sugar chain mapping or the like.
[0629] (1) Analysis of Neutral Sugar and Amino Sugar
Compositions
[0630] The composition analysis of the sugar chain of an antibody
molecule can be carried out by acid hydrolysis of sugar chains with
trifluoroacetic acid or the like to release a neutral sugar or an
amino sugar and measuring the composition ratio.
[0631] Examples include a method using a sugar composition analyzer
(BioLC) manufactured by Dionex. The BioLC is an apparatus which
analyzes a sugar composition by HPAEC-PAD (high performance
anion-exchange chromatography-pulsed amperometric detection) [J.
Liq. Chromatogr., 6, 1577 (1983)].
[0632] The composition ratio can also be analyzed by a fluorescence
labeling method using 2-aminopyridine. Specifically, the
composition ratio can be calculated in accordance with a known
method [Agric. Biol. Chem., 55, 283 (1991)] by labeling an
acid-hydrolyzed sample with a fluorescence by 2-aminopyridylation
and then analyzing the composition by HPLC.
[0633] (2) Analysis of Sugar Chain Structure
[0634] The sugar chain structure binding to an antibody molecule
can be analyzed by the two dimensional sugar chain mapping method
[Anal. Biochem., 171, 73 (1988), Biochemical Experimentation
Methods 23--Methods for Studying Glycoprotein Sugar Chains (Japan
Scientific Societies Press) edited by Reiko Takahashi (1989)]. The
two dimensional sugar chain mapping method is a method for deducing
a sugar chain structure by, e.g., plotting the retention time or
elution position of a sugar chain by reverse phase chromatography
as the X axis and the retention time or elution position of the
sugar chain by normal phase chromatography as the Y axis,
respectively, and comparing them with those of known sugar
chains.
[0635] Specifically, sugar chains are released from an antibody by
subjecting the antibody to hydrazinolysis, and the released sugar
chains are subjected to fluorescence labeling with 2-aminopyridine
(hereinafter referred to as "PA") [J. Biochem., 95, 197 (1984)],
and then the sugar chains are separated from an excess PA-treating
reagent by gel filtration, and subjected to reverse phase
chromatography. Thereafter, each peak of the separated sugar chains
are subjected to normal phase chromatography. From these results,
the sugar chain structure can be deduced by plotting the results on
a two dimensional sugar chain map and comparing them with the spots
of a sugar chain standard (manufactured by Takara Shuzo) or a
literature [Anal. Biochem., 171, 73 (1988)].
[0636] The structure deduced by the two dimensional sugar chain
mapping method can be confirmed by further carrying out mass
spectrometry such as MALDI-TOP-MS of each sugar chain.
[0637] 7. Immunological Determination Method for Identifying Sugar
Chain Structure of Antibody Molecule
[0638] An antibody composition comprises an antibody molecule in
which sugar chains binding to the Fc region of the antibody are
different in structure. The antibody composition comprising a sugar
chain in which fucose is not bound to N-acetylglucosamine in the
reducing end in the sugar chain among the total complex
N-glycoside-linked sugar chains binding to the Fc region in the
antibody composition reducing end can be identified by using the
method for analyzing the sugar chain structure binding to an
antibody molecule described in the item 6. Also, it can also be
identified by an immunological determination method using a
lectin.
[0639] The sugar chain structure binding to an antibody molecule
can be identified by the immunological determination method using a
lectin in accordance with the known immunological determination
method such as Western staining, IRA (radioimmunoassay), VIA
(viroimmunoassay), EIA (enzymoimmunoassay), FIA (fluoroimmunoassay)
or MIA (metalloimmunoassay) described in literatures [Monoclonal
Antibodies: Principles and Applications, Wiley-Liss, Inc. (1995);
Immunoassay (Koso Meneki Sokuteiho), 3rd Ed., Igakushoin (1987);
Revised Edition, Enzyme Antibody Method (Koso Kotaiho), Gakusai
Kikaku (11985)] and the like.
[0640] A lectin which recognizes the sugar chain structure binding
to an antibody molecule comprised in an antibody composition is
labeled, and the labeled lectin is allowed to react with a sample
antibody composition. Then, the amount of the complex of the
labeled lectin with the antibody molecule is measured.
[0641] The lectin used for identifying the sugar chain structure
binding to an antibody molecule includes WGA (wheat-germ agglutinin
derived from T. vulgaris), ConA (cocanavalin A derived from C.
ensiformis), RIC (toxin derived from R. communis), L-PHA
(leucoagglutinin derived from P. vulgaris), LCA (lentil agglutinin
derived from L. culinaris), PSA (pea lectin derived from P.
sativum), AAL (Aleuria aurantia lectin), ACL (Amaranthus caudatus
lectin), BPL (Bauhinia purpurea lectin), DSL (Datura stramonium
lectin), DBA (Dolichos biflorus agglutinin), EBL (elderberry balk
lectin), ECL (Erythrina cristagalli lectin), EEL (Euonymus
eoropaeus lectin), GNL (Galanthus nivalis lectin), GSL (Griffonia
simplicifolia lectin), HPA (Helix pomatia agglutinin), HM
(Hippeastrum hybrid lectin), Jacalin, LTL (Lotus tetragonolobus
lectin), LEL (Lycopersicon esculentum lectin), MAL (Maackia
amurensis lectin), MPL (Maclura pomifera lectin), NPL (Narcissus
pseudonarcissus lectin), PNA (peanut agglutinin), E-PHA (Phaseolus
vulgaris erythroagglutinin), PTL (Psophocarpus tetragonolobus
lectin), RCA (Ricinus communis agglutinin), STL (Solanum tuberosum
lectin), SIA (Sophora japonica agglutinin), SBA (soybean
agglutinin), UEA (Ulex europaeus agglutinin), VVL (Vicia villosa
lectin) and WFA (Wisteria floribunda agglutinin).
[0642] It is preferable to use a lectin which specifically
recognizes a sugar chain structure wherein fucose binds to the
N-acetylglucosamine in the reducing end in the complex
N-glycoside-linked sugar chain. Examples include Lens culinaris
lectin LCA (lentil agglutinin derived from Lens culinaris), pea
lectin PSA (pea lectin derived from Pisum sativum), broad bean
lectin VFA (agglutinin derived from Vicia faba) and Aleuria
aurantia lectin AAL (lectin derived from Aleuria aurantia).
[0643] 8. Method for Measuring Binding Activity of Antibody
Composition to Fc.gamma.RIIIa
[0644] The present invention relates to a method for detecting the
ratio of a sugar chain in which fucose is not bound to
N-acetylglucosamine in the reducing end in an antibody composition
by using a measuring method which comprises reacting an antigen
with a tested antibody composition to form a complex of the antigen
and the antibody composition; contacting the complex with an
Fc.gamma. receptor IIIa. Furthermore, the present invention relates
to a method for detecting the antibody-dependent cell-mediated
cytotoxic activity.
[0645] The measuring method used in the present invention is
described below in detail.
[0646] First, an antigen is fixed on a plate, and a sample of
antibody composition is allowed to react with it. The resulting
complex after the antigen-antibody reaction is allowed to react
with human Fc.gamma.IIIa.
[0647] The human Fc.gamma.RIIIa to be allowed to react is labeled
with a label such as an enzyme, a radioisotope or an fluorescent,
and the binding activity of the antibody bound to the antigen can
be measured by an immunological measuring method.
[0648] The immunological measuring method includes any method which
uses an antigen-antibody reaction such as an immunoassay, an
immunoblotting, a coagulation reaction, a complement binding
reaction, a hemolysis reaction, a precipitation reaction, a
colloidal gold method, a chromatography or an immune staining
method. Among these, the immunoassay is preferred.
[0649] Also, human Fc.gamma.RIIIa having a tag can be obtained by
ligating a nucleotide sequence encoding a short peptide to a gene
encoding the human Fc.gamma.RIIIa, and expressing the product by
genetic engineering techniques. The tag includes histidine and the
like.
[0650] Accordingly, when the above reaction is carried out by using
the human Fc.gamma.RIIIa having a tag, an immunoassay having high
sensitivity can be carried out by applying an antibody against the
tag after the reaction and labeling the antibody against the tag as
described above, or using a labeled antibody which binds to the
antibody against the tag.
[0651] Also, human Fc.gamma.RIIIa having a tag can be obtained by
ligating a nucleotide sequence encoding a short peptide to a gene
encoding the human Fc.gamma.RIIa, and expressing the product by
genetic engineering techniques. The tag includes histidine and the
like.
[0652] Accordingly, when the above reaction is carried out by using
the human Fc.gamma.RIIIa having a tag, an immunoassay having high
sensitivity can be carried out by applying an antibody against the
tag after the reaction and labeling the antibody against the tag as
described above, or using a labeled antibody which binds to the
antibody against the tag.
[0653] The detection method of the present invention can also be
carried out by directly contacting a sample of antibody composition
with Fc.gamma.RIIa, without reacting an antigen with the sample of
antibody component. For example, a labeled antibody which
recognizes the human Fc region can be detect ed by solid-phasing an
antibody against the tag and reacting the antibody with
tag-containing Fc.gamma.RIIIa, followed by reaction with a tested
antibody composition.
[0654] The method for detecting the ratio of a sugar chain in which
fucose is not bound to N-acetylglucosamine in the reducing end of
an antibody composition can be carried out according to the
following method.
[0655] First, antibody compositions (standards) necessary for the
preparation of a standard curve having a different ratio of a sugar
chain in which fucose is not bound to N-acetylglucaomine in the
sugar chain reducing end are prepared and the sugar chain analysis
of an antibody composition is carried out. In this case, the
concentrations of the antibody compositions are adjusted to the
same. Each of the binding activities of the prepared antibody
composition samples to Fc.gamma.RIIIa is measured, and a standard
curve between the ratio of sugar chain and the binding activity to
Fc.gamma.RIIa is prepared.
[0656] Based on the above standard curve, the binding activity of
the antibody composition sample to be measured to Fc.gamma.RIIIa is
measured while keeping the definite concentration of the antibody
composition according to a measuring method similar to the
above.
[0657] Based on the above standard curve, the binding activity of
the antibody composition to Fc.gamma.RIIIa is measured with the
same concentration of the sample to be measured according to the
measuring method similar to the above, so that the ratio of a sugar
chain in which fucose is not bound to N-acetylglucosamine in the
sugar chain reducing end in the antibody composition sample can be
obtained.
[0658] Furthermore, detection of ADCC activity can be carried out
by the following method.
[0659] ADCC activities of the standards used for the preparation of
the standard curve in the method for detecting the ratio of a sugar
chain in which fucose is not bound to N-acetylglucosamine in the
sugar chain reducing end of the above antibody composition are
measured. The measuring method includes the method for measuring
ADCC described below. Each of binding activities of the prepared
antibody composition samples is measured by the above measuring
method, and a standard curve between the ADCC activity and the
binding activity to Fc.gamma.RIIIa is prepared.
[0660] Based on the above standard curve, the binding activity of
the antibody composition sample to be measured to Fc.gamma.RIIa is
measured while keeping the definite concentration of the antibody
composition according to the measuring method used above, so that
the ADCC activity can be obtained.
[0661] 8 Screening Method for Antibody Composition
[0662] The present invention relates to a method for screening an
antibody composition having a higher binding activity to FcRIIIa,
which comprises reacting an antigen with a tested antibody,
followed by treatment with Fc.gamma.RIIIa.
[0663] The screening method of the present invention is described
below in detail.
[0664] An antigen is fixed on a plate and allowed to react with an
antibody to be tested. Human Fc.gamma.RIIIa is allowed to react
with the complex of the antigen-antibody reaction.
[0665] The human Fc.gamma.RIIIa to be allowed to react is labeled
with a label such as an enzyme, a radioisotope or an fluorescent,
and the binding activity of the antibody bound to the antigen can
be measured by an immunological measuring method.
[0666] The immunological measuring method includes any method which
uses an antigen-antibody reaction such as an immunoassay, an
immunoblotting, a coagulation reaction, a complement binding
reaction, a hemolysis reaction, a precipitation reaction, a
colloidal gold method, a chromatography or an immune staining
method. Among these, the immunoassay is preferred.
[0667] Also, human Fc.gamma.RIIIa having a tag can be obtained by
ligating a nucleotide sequence encoding a short peptide to a gene
encoding the human Fc.gamma.RIIIa, and expressing the product by
genetic engineering techniques. The tag includes histidine and the
like.
[0668] Accordingly, when the above reaction is carried out by using
the human Fc.gamma.RIIIa having a tag, an immunoassay having high
sensitivity can be carried out by applying an antibody against the
tag after the above reaction and labeling the antibody against the
tag as described above, or using a labeled antibody which binds to
the antibody against the tag.
[0669] 10. Application of Antibody Composition of the Present
Invention
[0670] The antibody composition obtained by the screening method of
the present invention has high ADCC activity.
[0671] An antibody having high ADCC activity is useful for
preventing and treating various diseases including cancers,
inflammatory diseases, immune diseases such as autoimmune diseases
and allergies, cardiovascular diseases and viral or bacterial
infections.
[0672] In the case of cancers, namely malignant tumors, cancer
cells grow. General anti-tumor agents are characterized by
inhibiting the growth of cancer cells. In contrast, an antibody
having high ADCC activity can treat cancers by injuring cancer
cells through its cell killing effect, and therefore, it is more
effective as a therapeutic agent than the general anti-tumor
agents. At present, in regard to therapeutic agents for cancers, an
anti-tumor effect of an antibody medicament alone is insufficient,
so that it has been taken to chemotherapy [Science, 280, 1197
(1998)]. If higher anti-tumor effect is found by the antibody
composition of the present invention alone, the dependency on
chemotherapy will be decreased and side effects will be
reduced.
[0673] In immune diseases such as inflammatory diseases, autoimmune
diseases and allergies, in vivo reactions of the diseases are
induced by the release of a mediator molecule by immunocytes, so
that the allergy reaction can be inhibited by eliminating
immunocytes using an antibody having high ADCC activity.
[0674] The cardiovascular diseases include arteriosclerosis and the
like. The arteriosclerosis is treated by using balloon catheter at
present, but cardiovascular diseases can be prevented and treated
by using an antibody having high ADCC activity because growth of
arterial cells in restructure after above treatment can be
inhibited by using the antibody.
[0675] Various diseases including viral and bacterial infections
can be prevented and treated by inhibiting proliferation of cells
infected with a virus or bacterium using an antibody having high
ADCC activity.
[0676] Specific examples of an antibody which recognizes a
tumor-related antigen, an antibody which recognizes an allergy- or
inflammation-related antigen, an antibody which recognizes
cardiovascular disease-related antigen and an antibody which
recognizes a viral or bacterial infection-related antigen are
described below.
[0677] The antibody which recognizes a tumor-related antigen
includes anti-GD2 antibody [Anticancer Res., 13, 331 (1993)],
anti-GD3 antibody [Cancer Immunol. Immunother., 36, 260 (1993)],
anti-GM2 antibody [Cancer Res., 54, 1511 (1994)], anti-HER2
antibody [Proc. Natl. Acad. Sci. USA, 89, 4285 (1992)], anti-CD52
antibody [Proc. Natl. Acad. Sci. USA, 89, 4285 (1992)], anti-MAGE
antibody [British J. Cancer, 83, 493 (2000)], anti-HM1.24 antibody
[Molecular Immunol., 36, 387 (1999)], anti-parathyroid
hormone-related protein (PTHrP) antibody [Cancer, 88, 2909 (2000)],
anti-basic fibroblast growth factor antibody and anti-FGF8 antibody
[Proc. Natl. Acad. Sci. USA, 86, 9911 (1989)], anti-basic
fibroblast growth factor receptor antibody and anti-FGF8 receptor
antibody [J. Biol. Chem., 265, 16455 (1990)], anti-insulin-like
growth factor antibody [J. Neurosci. Res., 40, 647 (1995)],
anti-insulin-like growth factor receptor antibody [J. Neurosci.
Res., 40, 647 (1995)], anti-PMSA antibody [J. Urology, 160, 2396
(1998)], anti-vascular endothelial cell growth factor antibody
[Cancer Res., 57, 4593 (1997)], anti-vascular endothelial cell
growth factor receptor antibody [Oncogene, 19, 2138 (2000)] and the
like.
[0678] The antibody which recognizes an allergy- or
inflammation-related antigen includes anti-interleukin 6 antibody
[Immunol. Rev, 127, 5 (1992)], anti-interleukin 6 receptor antibody
[Molecular Immunol., 31, 371 (1994)], anti-interleukin 5 antibody
[Immunol. Rev., 127, 5 (1992)], anti-interleukin 5 receptor
antibody and anti-interleukin 4 antibody [Cytokine, 3, 562 (1991)],
anti-interleukin 4 receptor antibody [J. Immunol. Methods, 217, 41
(1998)], anti-tumor necrosis factor antibody [Hybridoma, 13, 183
(1994)], anti-tumor necrosis factor receptor antibody [Molecular
Pharmacol., 58, 237(2000)], anti-CCR4 antibody [Nature, 400, 776
(1999)], anti-chemokine antibody [J. Immuno. Meth., 174, 249
(1994)], anti-chemokine receptor antibody [J. Exp. Med., 186, 1373
(1997)] and the like. The antibody which recognizes a
cardiovascular disease-related antigen includes anti-GpIIb/IIIa
antibody [J. Immunol., 152, 2968 (1994)], anti-platelet-derived
growth factor antibody [Science, 253, 1129 (1991)],
anti-platelet-derived growth factor receptor antibody [J. Biol.
Chem., 272, 17400 (1997)] and anti-blood coagulation factor
antibody [Circulation, 101, 1158 (2000)] and the like.
[0679] The antibody which recognizes a viral or bacterial
infection-related antigen includes anti-gp120 antibody [Structure,
8, 385 (2000)], anti-CD4 antibody [J. Rheumatology, 25, 2065
(1998)], anti-CCR5 antibody and anti-Vero toxin antibody [J. Clin.
Microbiol., 37, 396 (1999)] and the like.
[0680] These antibodies can be obtained from public organizations
such as ATCC (The American Type Culture Collection), RIKEN Gene
Bank at The Institute of Physical and Chemical Research and
National Institute of Bioscience and Human Technology, Agency of
Industrial Science and Technology, or private reagent sales
companies such as Dainippon Pharmaceutical, R & D SYSTEMS,
PharMingen, Cosmo Bio and Funakoshi Co., Ltd.
[0681] The antibody composition obtained by the process of the
present invention can be administered as various therapeutic agents
alone, but generally, it is preferable to provide it as a
pharmaceutical formulation produced by an appropriate method well
known in the technical field of pharmaceutical, by mixing it with
one or more pharmaceutically acceptable carriers.
[0682] It is preferable to select a route of administration which
is most effective in treatment. Examples include oral
administration and parenteral administration, such as buccal,
tracheal, rectal, subcutaneous, intramuscular and intravenous. In
the case of an antibody preparation, intravenous administration is
preferred.
[0683] The dosage form includes sprays, capsules, tablets,
granules, syrups, emulsions, suppositories, injections, ointments,
tapes and the like.
[0684] The pharmaceutical preparation suitable for oral
administration include emulsions, syrups, capsules, tablets,
powders, granules and the like.
[0685] Liquid preparations such as emulsions and syrups can be
produced by using, as additives, water; sugars such as sucrose,
sorbitol and fructose, glycols such as polyethylene glycol and
propylene glycol, oils such as sesame oil, olive oil and soybean
oil; antiseptics such as p-hydroxybenzoic acid esters; flavors such
as strawberry flavor and peppermint; and the like.
[0686] Capsules, tablets, powders, granules and the like can be
prepared by using, as additives, excipients such as lactose,
glucose, sucrose and mannitol; disintegrating agents such as starch
and sodium alginate; lubricants such as magnesium stearate and
talc; binders such as polyvinyl alcohol, hydroxypropylcellulose and
gelatin; surfactants such as fatty acid ester; plasticizers such as
glycerine; and the like.
[0687] The pharmaceutical preparation suitable for parenteral
administration includes injections, suppositories, sprays and the
like.
[0688] Injections may be prepared by using a carrier such as a salt
solution, a glucose solution or a mixture thereof. Also, powdered
injections can be prepared by freeze-drying the antibody
composition in the usual way and adding sodium chloride
thereto.
[0689] Suppositories may be prepared by using a carrier such as
cacao butter, hydrogenated fat or carboxylic acid.
[0690] Also, sprays may be prepared by using the antibody
composition as such or using a carrier, etc. which do not stimulate
the buccal or airway mucous membrane of the patient and can
facilitate absorption of the antibody composition by dispersing it
as fine particles.
[0691] The carrier includes lactose, glycerine and the like.
Depending on the properties of the antibody composition and the
carrier, it is possible to produce pharmaceutical preparations such
as aerosols and dry powders. In addition, the components
exemplified as additives for oral preparations can also be added to
the parenteral preparations.
[0692] Although the clinical dose or the frequency of
administration varies depending on the objective therapeutic
effect, administration method, treating period, age, body weight
and the like, it is usually 10 .mu.g/kg to 20 mg/kg per day and per
adult.
[0693] Also, as the method for examining antitumor effect of the
antibody composition against various tumor cells, in vitro tests
include CDC activity measuring method, ADCC activity measuring
method and the like, and in vivo tests include antitumor
experiments using a tumor system in an experimental animal such as
a mouse, and the like.
[0694] CDC activity and ADCC activity measurements and antitumor
experiments can be carried out in accordance with the methods
described in Cancer Immunology Immunotherapy, 36, 373 (1993);
Cancer Research, 54, 1511 (1994) and the like.
[0695] The present invention will be described below in detail
based on Examples; however, Examples are only simple illustrations,
and the scope of the present invention is not limited thereto.
BEST MODE FOR CARRYING OUT THE INVENTION
EXAMPLE 1
[0696] Preparation of Anti-Ganglioside GD3 Human Chimeric
Antibody:
[0697] 1. Construction of Tandem Expression Vector pChi641LHGM4 for
Anti-Ganglioside GD3 Human Chimeric Antibody
[0698] A plasmid pChi641LGM40 was constructed by ligating a
fragment of about 4.03 kb containing an L chain cDNA, obtained by
digesting an L chain expression vector pChi641LGM4 [J. Immunol.
Methods, 167, 271 (1994)] for anti-ganglioside GD3 human chimeric
antibody (hereinafter referred to as "anti-GD3 chimeric antibody")
with restriction enzymes MluI (manufactured by Takara Shuzo) and
SalI (manufactured by Takara Shuzo) with a fragment of about 3.40
kb containing a G418-resistant gene and a splicing signal, obtained
by digesting an expression vector pAGE107 [Cytotechnology, 3, 133
(1990)] for animal cell with restriction enzymes MluI (manufactured
by Takara Shuzo) and SalI (manufactured by Takara Shuzo) using DNA
Ligation Kit (manufactured by Takara Shuzo), and then transforming
E. coli HB101 (Molecular Cloning, Second Edition) with the ligated
product.
[0699] Next, a fragment of about 5.68 kb containing an L chain
cDNA, obtained by digesting the constructed plasmid pChi641LGM40
with a restriction enzyme ClaI (manufactured by Takara Shuzo),
changing it to blunt-end using DNA Blunting Kit (manufactured by
Takara Shuzo) and further digesting it with MluI (manufactured by
Takara Shuzo), was ligated with a fragment of about 8.40 kb
containing an H chain cDNA, obtained by digesting an anti-GD3
chimeric antibody H chain expression vector pChi641GM4 [J. Immunol.
Methods, 1, 271 (1994)] with a restriction enzyme (manufactured by
Takara Shuzo), changing it to blunt-end using DNA Blunting Kit
(manufactured by Takara Shuzo) and further digesting it with MluI
(manufactured by Takara Shuzo) using DNA Ligation Kit (manufactured
by Takara Shuzo), and then E. coli HB101 (Molecular Cloning, Second
Edition) was transformed with the ligated product to thereby
construct a tandem expression vector pChi641LHGM4 for anti-GD3
chimeric antibody.
[0700] 2. Preparation of Cell Stably Producing Anti-GD3 Chimeric
Antibody
[0701] Cells capable of stably producing an anti-GD3 chimeric
antibody were prepared by introducing the tandem expression vector
pChi641LHGM4 for anti-GD3 chimeric antibody constructed in the item
1 of Example 1 and selecting suitable clones, as described
below.
[0702] (1) Preparation of Producing Cell Using Rat Myeloma YB2/0
Cell
[0703] After introducing 5 .mu.g of the anti-GD3 chimeric antibody
expression vector pChi641LHGM4 into 4.times.10.sup.6 cells of rat
myeloma YB2/0 [ATCC CRL-1662, J. Cell. Biol., 93, 576 (1982)] by
electroporation [Cytotechnology, 3, 133 (1990)], the cells were
suspended in 40 ml of RPMI1640-FBS(10) (RPMI1640 medium comprising
10% (fetal bovine serum (hereinafter referred to as "FBS")
(manufactured by GIBCO BRL)) and dispensed at 200 .mu.l/well into a
96 well culture plate (manufactured by Sumitomo Bakelite). After
culturing at 37.degree. C. for 24 hours in a 5% CO.sub.2 incubator,
G418 was added to give a concentration of 0.5 mg/ml, followed by
culturing for 1 to 2 weeks. The culture supernatant was recovered
from wells in which colonies of transformants showing G418
resistance were formed and growth of colonies was observed, and the
antigen binding activity of the anti-GD3 chimeric antibody in the
supernatant was measured by the ELISA shown in the item 3 of
Example 1.
[0704] Regarding the transformants in wells in which production of
the anti-GD3 chimeric antibody was observed in culture
supernatants, in order to increase the amount of the antibody
production using a DHFR gene amplification system, each of them was
suspended in the RPMI1640-FBS(10) medium comprising 0.5 mg/ml G418
and 50 nmol/L DHFR inhibitor, methotrexate (hereinafter referred to
as "MTX"; manufactured by SIGMA) to give a density of 1 to
2.times.10.sup.5 cells/ml, and the suspension was dispensed at 2 ml
into each well of a 24 well plate (manufactured by Greiner).
Transformants showing 50 nmol/L MTX resistance were induced by
culturing at 37.degree. C. for 1 to 2 weeks in a 5% CO.sub.2
incubator. The antigen binding activity of the anti-GD3 chimeric
antibody in culture supernatants in wells in which growth of
transformants was observed was measured by the ELISA shown in the
item 3 of Example 1. Regarding the transformants in wells in which
production of the anti-GD3 chimeric antibody was observed in
culture supernatants, the MTX concentration was increased to 100
nmol/L and then to 200 nmol/L, and transformants capable of growing
in the RPMI1640-FBS(10) medium comprising 0.5 mg/ml G418 and 200
nmol/L MTX and capable of producing the anti-GD3 chimeric antibody
in a large amount were finally obtained by the same method as
described above. Among the obtained transformants, suitable clones
were selected and were made into a single cell (cloning) by
limiting dilution twice.
[0705] The obtained anti-GD3 chimeric antibody-producing trans
formed cell clone 7-9-51 has been deposited on Apr. 5, 1999, as
FERM BP-6691 in National Institute of Bioscience and Human
Technology, Agency of Industrial Science and Technology (Higashi
1-1-3, Tsukuba, Ibaraki, Japan) (present name: International Patent
Organism Depositary, National Institute of Advanced Industrial
Science and Technology (Tsukuba Central 6, 1, Higashi 1-Chome
Tsukuba-shi, Ibaraki-ken, Japan)).
[0706] (2) Preparation of Producing Cell Using CHO/DG44 Cell
[0707] After introducing 4 .mu.g of the anti-GD3 chimeric antibody
expression vector pChi641LHGM4 into 1.6.times.10.sup.6 cells of
CHO/DG44 cell [Proc. Natl. Acad. Sci. USA, 77, 4216 (1980)] by
electroporation [Cytotechnology, 3, 133 (1990)], the cells were
suspended in 10 ml of IMDM-FBS(10)-HT(1) [IMDM medium comprising
10% FBS and 1.times. concentration of HT supplement (manufactured
by GIBCO BRL)] and dispensed at 200 .mu.l/well into a 96 well
culture plate (manufactured by Iwaki Glass). After culturing at
37.degree. C. for 24 hours in a 5% CO.sub.2 incubator, G418 was
added to give a concentration of 0.5 mg/ml, followed by culturing
for 1 to 2 weeks. The culture supernatant was recovered from wells
in which colonies of transformants showing G418 resistance were
formed and growth of colonies was observed, and the antigen binding
activity of the anti-GD3 chimeric antibody in the supernatant was
measured by the ELISA shown in the item 3 of Example 1.
[0708] Regarding the transformants in wells in which production of
the anti-GD3 chimeric antibody was observed in culture
supernatants, in order to increase the amount of the antibody
production using a DHFR gene amplification system, each of them was
suspended in an IMDM-dFBS(10) medium [IMDM medium comprising 10%
dialyzed fetal bovine serum (hereinafter referred to as "dFBS";
manufactured by GIBCO BRL)] comprising 0.5 mg/ml G418 and 10 nmol/L
MTX to give a density of 1 to 2.times.10.sup.5 cells/ml, and the
suspension was dispensed at 0.5 ml into each well of a 24 well
plate (manufactured by Iwaki Glass). Transformants showing 10
nmol/L MTX resistance were induced by culturing at 37.degree. C.
for 1 to 2 weeks in a 5% CO.sub.2 incubator. Regarding the
transformants in wells in which their growth was observed, the MTX
concentration was increased to 100 nmol/L, and transformants
capable of growing in the IMDM-dFBS(10) medium comprising 0.5 mg/ml
G418 and 100 nmol/L MTX and of producing the anti-GD3 chimeric
antibody in a large amount were finally obtained by the same method
as described above. Among the obtained transformants, suitable
clones were selected and were made into a single cell (cloning) by
limiting dilution twice.
[0709] (3) Preparation of Producing Cell Using Mouse Myeloma NS0
Cell
[0710] After introducing 5 .mu.g of the anti-GD3 chimeric antibody
expression vector pChi641LHGM4 into 4.times.10.sup.6 cells of mouse
myeloma NS0 by electroporation [Cytotechnology, 3, 133 (1990)], the
cells were suspended in 40 ml of EX-CELL302-FBS(10) (EX-CELL302
medium comprising 10% FBS and 2 mmol/L L-glutamine (hereinafter
referred to as "L-Gln"; manufactured by GIBCO BPL) and dispensed at
200 .mu.l/well into a 96 well culture plate (manufactured by
Sumitomo Bakelite). After culturing at 37.degree. C. for 24 hours
in a 5% CO.sub.2 incubator, G418 was added to give a concentration
of 0.5 mg/ml, followed by culturing for 1 to 2 weeks. The culture
supernatant was recovered from wells in which colonies of
transformants showing G418 resistance were formed and growth of
colonies was observed, and the antigen binding activity of the
anti-GD3 chimeric antibody in the supernatant was measured by the
ELISA shown in the item 3 of Example 1.
[0711] Regarding the transformants in wells in which production of
the anti-GD3 chimeric antibody was observed in culture
supernatants, in order to increase the amount of the antibody
production using a DHFR gene amplification system, each of them was
suspended in an EX-CELL302-dFBS(10) medium (EX-CELL302 medium
comprising 10% dFBS and 2 mmol/L L-Gln) comprising 0.5 mg/ml G418
and 50 nmol/L MTX to give a density of 1 to 2.times.10.sup.5
cells/ml, and the suspension was dispensed at 2 ml into each well
of a 24 well plate (manufactured by Greiner). Transform ants
showing 50 nmol/L MTX resistance were induced by culturing at
37.degree. C. for 1 to 2 weeks in a 5% CO.sub.2 incubator. The
antigen binding activity of the anti-GD3 chimeric antibody in
culture supernatants in wells in which growth of transformants was
observed was measured by the ELISA shown in the item 3 of Example
1. Regarding the transformants in wells in which production of the
anti-GD3 chimeric antibody was observed in culture supernatants,
the MTX concentration was increased to 100 nmol/L and then to 200
mmol/L, and transformants capable of growing in the
EX-CELL302-dFBS(10) medium comprising 0.5 mg/ml G418 and 200 nmol/L
MTX and of producing the anti-GD3 chimeric antibody in a large
amount was finally obtained by the same method as described above.
Among the obtained transformants, suitable clones were selected and
were made into a single cell (cloning) by limiting dilution twice.
Also, using the method for determining the transcription product of
an .alpha.1,6-fucosyltransferase gene shown in Example 9, a clone
producing a relatively small amount of the transcription product
was selected and used as a suitable clone.
[0712] 3. Measurement of Binding Activity of Antibody to GD3
(ELISA)
[0713] The binding activity of the antibody to GD3 was measured as
described below.
[0714] In 2 ml of an ethanol solution containing 10 .mu.g of
dipalmitoylphosphatidylcholine (manufactured by SIGMA) and 5 .mu.g
of cholesterol (manufactured by SIGMA), 4 nmol of GD3 (manufactured
by Snow Brand Milk Products) was dissolved. Into each well of a 96
well plate for ELISA (manufactured by Greiner), 20 .mu.l of the
solution (40 pmol/well in final concentration) was dispensed,
followed by air-drying, 1% bovine serum albumin (hereinafter
referred to as "S" manufactured by SIGMA)-containing PBS
(hereinafter referred to as "1% BSA-PBS") was dispensed at 100
.mu.l/well, and then the reaction was carried out at room
temperature for 1 hour to block remaining active groups. After
discarding 1% BSA-PBS, a culture supernatant of a transformant or a
diluted solution of a human chimeric antibody was dispensed at 50
.mu.l/well to carry out the reaction at room temperature for 1
hour. After the reaction, each well was washed with 0.05% Tween 20
(manufactured by Wako Pure Chemical Industries)-containing PBS
(hereinafter referred to as "Tween-PBS"), a peroxidase-labeled goat
anti-human IgG (H & L) antibody solution (manufactured by
American Qualex) diluted 3,000 times with 1% BSA-PBS was dispensed
at 50 .mu.l/well as a secondary antibody solution, and then the
reaction was carried out at room temperature for 1 hour. After the
reaction and subsequent washing with Tween-PBS, ABTS substrate
solution [solution prepared by dissolving 0.55 g of
2,2'-azino-bis(3-ethylbenzothiazoline-6-- sulfonic acid)ammonium
salt in 1 liter of 0.1 mol/L citrate buffer (pH 4.2) and adding 1
.mu.l/ml of hydrogen peroxide to the solution just before use
(hereinafter the same solution was used)] was dispensed at 50
.mu.l/well for color development, and then absorbance at 415 nm
(hereinafter referred to as "OD415") was measured.
[0715] 4. Purification of Anti-GD3 Chimeric Antibody
[0716] (1) Culturing of Producing Cell Derived from YB 2/0 Cell and
Purification of Antibody
[0717] The anti-GD3 chimeric antibody-producing transformed cell
clone obtained the item 2(1) of Example 1 was suspended in the
Hybridoma-SFM medium comprising 0.2% BSA, 200 nmol/L MTX and 100
nmol/L triiodothyronine (hereinafter referred to as "T3";
manufactured by SIGMA) to give a density of 3.times.10.sup.5
cells/ml and cultured in a 2.0 liter bottle (manufactured by Iwaki
Glass) under stirring at a rate of 50 rpm. After culturing at
37.degree. C. for 10 days in a temperature-controlling room, the
culture supernatant was recovered. The anti-GD3 chimeric antibody
was purified from the culture supernatant using a Prosep-A
(manufactured by Bioprocessing) column in accordance with the
manufacture's instructions. The purified anti-GD3 chimeric antibody
was named YB2/0-GD3 chimeric antibody.
[0718] (2) Culturing of Producing Cell Derived from CHO/DG44 Cell
and Purification of Antibody
[0719] The anti-GD3 chimeric antibody-producing transformed cell
clone obtained in the item 2(2) of Example 1 was suspended in the
EX-CELL302 medium comprising 3 mmol/L L-Gln, 0.5% fatty acid
concentrated solution (hereinafter referred to as "CDLC";
manufactured by GIBCO BRL) and 0.3% Pluronic F68 (hereinafter
referred to as "PF68"; manufactured by GIBCO BRL) to give a density
of 1.times.10.sup.6 cells/ml, and the suspension was dispensed at
50 ml into 175 mm.sup.2 flasks (manufactured by Greiner). After
culturing at 37.degree. C. for 4 days in a 5% CO.sub.2 incubator,
the culture supernatant was recovered. The anti-GD3 chimeric
antibody was purified from the culture supernatant using a Prosep-A
(manufactured by Bioprocessing) column in accordance with the
manufacture's instructions. The purified anti-GD3 chimeric antibody
was named CHO/DG44-GD3 chimeric antibody.
[0720] (3) Culturing of Producing Cell Derived from NS0 Cell and
Purification of Antibody
[0721] The anti-GD3 chimeric antibody-producing transformed cell
clone obtained in the item 2(3) of Example 1 was suspended in the
EX-CELL302 medium comprising 2 mmol/L L-Gln, 0.5 mg/ml G418, 200
nmol/L MTX and 1% FBS, to give a density of 1.times.10.sup.6
cells/ml, and the suspension was dispensed at 200 ml into 175
mm.sup.2 flasks (manufactured by Greiner). After culturing at
37.degree. C. for 4 days in a 5% CO.sub.2 incubator, the culture
supernatant was recovered. The anti-GD3 chimeric antibody was
purified from the culture supernatant using a Prosep-A
(manufactured by Bioprocessing) column in accordance with the
manufacture's instructions. The purified anti-GD3 chimeric antibody
was named NS0-GD3 chimeric antibody (302).
[0722] Also, the transformed cell clone was suspended in the GIT
medium comprising 0.5 mg/ml G418 and 200 nmol/L MTX to give a
density of 3.times.10: cells/ml, and the suspension was dispensed
at 200 ml into 175 mm.sup.2 flasks (manufactured by Greiner). After
culturing at 37.degree. C. for 10 days in a 5% CO.sub.2 incubator,
the culture supernatant was recovered. The anti-GD3 chimeric
antibody was purified from the culture supernatant using a Prosep-A
(manufactured by Bioprocessing) column in accordance with the
manufacture's instructions. The purified anti-GD3 chimeric antibody
was named NS0-GD3 chimeric antibody (GIT).
[0723] (4) Culturing of Producing Cell Derived from SP2/0 Cell and
Purification of Antibody
[0724] The anti-GD3 chimeric antibody-producing transformed cell
clone (KM-871 (FERM BP-3512)) described in Japanese Published
Unexamined Patent Application No. 304989/93 (EP 533199) was
suspended in the GIT medium comprising 0.5 mg/ml G418 and 200
nmol/L MTX to give a density of 3.times.10.sup.5 cells/ml, and the
suspension was dispensed at 200 ml into 175 mm.sup.2 flasks
(manufactured by Greiner). After culturing at 37.degree. C. for 8
days in a 5% C07 incubator, the culture supernatant was recovered.
The anti-CD3 chimeric antibody was purified from the culture
supernatant using a Prosep-A (manufactured by Bioprocessing) column
in accordance with the manufacture's instructions. The purified
anti-GD3 chimeric antibody was named SP2/0-GD3 chimeric
antibody.
[0725] 5. Analysis of Purified Anti-GD3 Chimeric Antibody
[0726] In accordance with a known method [Nature, 227, 680 (1970)],
4 .mu.g of each of the five kinds of the anti-GD3 chimeric
antibodies produced by and purified from respective animal cells,
obtained in the item 4 of Example 1, was subjected to SDS-PAGE to
analyze the molecular weight and purity. The results are shown in
FIG. 1. As shown in FIG. 1, a single band of about 150 kilodaltons
(hereinafter referred to as "Kd") in molecular weight was found
under non-educing conditions, and two bands of about 50 Kd and
about 25 Kd under reducing conditions, in each of the purified
anti-GD3 chimeric antibodies. The molecular weights almost
coincided with the molecular weights deduced from the cDNA
nucleotide sequences of H chain and L chain of the antibody (H
chain: about 49 Kd, L chain: about 23 Kd, whole molecule: about 144
Kd), and also coincided with the reports rating that the IgG
antibody has a molecular weight of about 150 Kd under non-reducing
conditions and is degraded into H chains having a molecular weight
of about 50 Kd and L chains having a molecular weight of about 25
Kd under reducing conditions due to cutting of the disulfide bond
(hereinafter referred to as "S--S bond") in the molecule
(Antibodies, Chapter 14; Monoclonal Antibodies), so that it was
confirmed that each anti-GD3 chimeric antibody was expressed and
purified as an antibody molecule having the true structure.
EXAMPLE 2
[0727] Activity Evaluation of Anti-GD3 Chimeric Antibody:
[0728] 1. Binding Activity of Anti-GD3 Chimeric Antibody to GD3
(ELISA)
[0729] Binding activities of the five kinds of the purified
anti-GD3 chimeric antibodies obtained in the item 4 of Example 1 to
GD3 were measured by the ELISA shown in the item 3 of Example 1.
FIG. 2 shows results of the examination of the binding activity
measured by changing the concentration of the anti-GD3 chimeric
antibody to be added. As shown in FIG. 2, the five kinds of the
anti-GD3 chimeric antibodies showed almost the same binding
activity to GD3. The result shows that antigen binding activities
of these antibodies are constant independently of the
antibody-producing animal cells and their culturing methods. Also,
it was suggested from the comparison of the NS0-GD3 chimeric
antibody (302) with the NS0-GD3 chimeric antibody (GIT) that the
antigen binding activities are constant independently of the media
used in the culturing.
[0730] 2. ADCC Activity of Anti-GD3 Chimeric Antibody
[0731] ADCC activities of the five kinds of the purified anti-GD3
chimeric antibodies obtained in the item 4 of Example 1 were
measured in accordance with the following method.
[0732] (1) Preparation of Target Cell Solution
[0733] A human melanoma cell line G-361 (ATCC CRL 1424) was
cultured in the RPMI1640-FBS(10) medium to prepare 1.times.10.sup.6
cells, and the cells were radioisotope-labeled by reacting them
with 3.7 MBq equivalents of a radioactive substance
Na.sub.2.sup.51CrO.sub.4 at 37.degree. C. for 1 hour. After the
reaction, the cells were washed three times through their
suspension in the RPMI1640-FBS(10) medium and centrifugation,
re-suspended in the medium and then allowed to react at 4.degree.
C. for 30 minutes on ice for spontaneous dissolution of the
radioactive substance. After centrifugation, the precipitate was
adjusted to 2.times.10.sup.5 cells/ml by adding 5 ml of the
RPMI640-FBS(10) medium and used as the target cell solution.
[0734] (2) Preparation of Effector Cell Solution
[0735] From a healthy doner, 50 ml of venous blood was collected,
and gently mixed with 0.5 ml of heparin sodium (manufactured by
Takeda Pharmaceutical). The mixture was centrifuged to isolate a
mononuclear cell layer using Lymphoprep (manufactured by Nycomed
Pharma AS) in accordance with the manufacture's instructions. After
washing with the RPMI1640-FBS(10) medium by centrifugation three
times, the resulting precipitate was re-suspended to give a density
of 2.times.10.sup.6 cells/ml by using the medium and used as the
effector cell solution.
[0736] (3) Measurement of ADCC Activity
[0737] Into each well of a 96 well U-shaped bottom plate
(manufactured by Falcon), 50 .mu.l of the target cell solution
prepared in the above (1) (1.times.10.sup.4 cells/well) was
dispensed. Next, 100 .mu.l of the effector cell solution prepared
in the above (2) was added thereto (2.times.10.sup.5 cells/well,
the ratio of effector cells to target cells becomes 20:1).
Subsequently, each of the anti-GD3 chimeric antibodies was added at
various concentrations, followed by reaction at 37.degree. C. for 4
hours. After the reaction, the plate was centrifuged, and the
amount of .sup.51Cr in the supernatant was measured with a
.gamma.-counter. The amount of spontaneously released .sup.51Cr was
calculated by the same operation using only the medium instead of
the effector cell solution and the antibody solution, and measuring
the amount of .sup.51Cr in the supernatant. The amount of total
released .sup.51Cr was calculated by the same operation as above
using only the medium instead of the antibody solution and adding 1
N hydrochloric acid instead of the effector cell solution, and
measuring the amount of .sup.51Cr in the supernatant. The ADCC
activity was calculated from the following equation (I). 1 ADCC
activity ( % ) = 51 Cr in sample supernatant - spontaneously
released 51 Cr total released 51 Cr - spontaneously released 51 Cr
.times. 100 ( 1 )
[0738] The results are shown in FIG. 3. As shown in FIG. 3, among
the five kinds of the anti-GD3 chimeric antibodies, the YB2/0-GD3
chimeric antibody showed the highest ADCC activity, followed by the
SP2/0-GD3 chimeric antibody, NS0-GD3 chimeric antibody and CHO-GD3
chimeric ant-body in that order. No difference in the ADCC activity
was found between the NS0-GD3 chimeric antibody (302) and NS0-GD3
chimeric antibody (GIT) prepared by using different media in the
culturing. The above results show that the ADCC activity of
antibodies greatly varies depending on the kind of the animal cells
to be used in their production. As its mechanism, since their
antigen binding activities were equal, it was considered that ADCC
activity depends on a difference in the structure of the Fc region
of the antibody.
EXAMPLE 3
[0739] Activity Evaluation of Anti-GD3 Chimeric Antibodies Having a
Different Ratio of a Sugar Chain in which 1-Position of Fucose is
not Bound to 6-Position of N-acetylglucosamine in the Reducing
End:
[0740] 1. Preparation of Anti-GD3 Chimeric Antibodies Having a
Different Ratio of a Sugar Chain in which 1-Position of Fucose is
not Bound to 6-Position of N-acetylglucosamine in the Reducing End
through .alpha.-Bond
[0741] In accordance with the method described in the item 2(1) of
Example 1, some transformed clones derived from YB2/0 cell capable
of producing an anti-GD3 chimeric antibody was obtained. Antibodies
were prepared from the transformed clones derived from YB2/0 cell
and named lot 1, lot 2 and lot 3. Sugar chain analysis of the
anti-GD3 chimeric antibodies of lot 1, lot 2 and lot 3 was carried
out by the following method.
[0742] The solution of ea ch purified antibody was exchanged to 10
mmol/L KH.sub.2PO.sub.4 using Ultra Free 0.5-10K (manufactured by
Millipore). The exchange was carried out in such a manner that the
exchanging ratio became 80-fold or more.
[0743] Into Hydraclub S-204 test tube, 100 .mu.g of each antibody
was put and dried with a centrifugal evaporator. The dried sample
was subjected to hydrazinolysis using Hydraclub manufactured by
Hohnen. The sample was allowed to react with hydrazine at
110.degree. C. for 1 hour using a hydrazinolysis reagent
manufactured by Hohnen [Method of Enzymology, 83, 263 (1982)].
After the reaction, hydrazine was evaporated under a reduced
pressure, and the reaction tube was returned to room temperature by
allowing it to stand for 30 minutes. Next, 250 of an acetylation re
agent manufactured by Hohnen and 25 .mu.l of acetic anhydride were
added thereto, followed by thoroughly stirred for reaction at room
temperature for 30 minutes. Then, 250 .mu.l of the acetylation
reagent and 25 .mu.l of acetic anhydride were further added
thereto, followed by thoroughly stirring for reaction at room
temperature for 1 hour. The sample was frozen at -80.degree. C. in
a freezer and freeze-dried for about 17 hours. Sugar chains were
recovered from the freeze-dried sample by using Cellulose Cartridge
Glycan Preparation Kit manufactured by Takara Shuzo. The sample
sugar chain solution was dried with a centrifugal evaporator and
then subjected to fluorescence labeling with 2-aminopyridine [J.
Biochem., 95, 197 (1984)]. The 2-aminopyridine solution was
prepared by adding 760 .mu.l of HCl per 1 g of 2-aminopyridine
(1.times.PA solution) and diluting the solution 10-fold with
reverse osmosis purified water (10-fold diluted PA solution). The
sodium cyanoborohydride solution was prepared by adding 20 .mu.l of
1.times.PA solution and 430 .mu.l of reverse osmosis purified water
per 10 mg of sodium cyanoborohydride. To the sample, 67 lt of a 10
fold-diluted PA solution was added, followed by reaction at
100.degree. C. for 15 minutes and spontaneously cooled, and 2 .mu.l
of sodium cyanoborohydride was further added thereto, followed by
reaction at 90.degree. C. for 12 hours for fluorescence labeling of
the sample sugar chains. The fluorescence-labeled sugar chain group
(PA-treated sugar chain group) was separated from excess reagent by
using Superdex Peptide HR 10/30 column (manufactured by Pharmacia).
This step was carried out by using 10 mmol/L ammonium bicarbonate
as the eluent at a flow rate of 0.5 ml/ml and at a column
temperature of room temperature, and using a fluorescence detector
of 320 nm excitation wavelength and 400 nm fluorescence wavelength.
The eluate was recovered 20 to 30 minutes after addition of the
sample and dried with a centrifugal evaporator to be used as
purified PA-treated sugar chains.
[0744] Next, reverse phase HPLC analysis of the purified PA-treated
sugar chains was carried out by using CLC-ODS column (manufactured
by Shimadzu, .phi. 6.0 nm.times.159 nm). The step was carried out
at a column temperature of 55.degree. C. and at a flow rate of 1
ml/min and using a fluorescence detector of 320 nm excitation
wavelength and 400 nm fluorescence wavelength. The column was
equilibrated with a 10 mmol/L sodium phosphate buffer (pH 3.8) and
elution was carried out for 80 minutes by a 0.5% 1-butanol linear
density gradient. FIG. 4 shows elution patterns of the purified
PA-treated sugar chains of the anti-GD3 antibody of lot 2. Each of
the PA-treated sugar chain was identified by post source decay
analysis of each peak of the separated PA-treated sugar chains
using matrix-assisted laser ionization type of flight mass
spectrometry (MALDI-TOF-MS analysis), comparison of elution
positions with standards of PA-treated sugar chain manufactured by
Takara Shuzo, and reverse phase HPLC analysis after digestion of
each PA-treated sugar chain using various enzymes.
[0745] The sugar chain content was calculated from each of the peak
area of PA-treated sugar chain by reverse HPLC analysis. A
PA-treated sugar chain whose reducing end is not
N-acetylglucosamine was excluded from the peak area calculation,
because it is an impurity or a by-product during preparation of
PA-treated sugar chain. Peaks (i) to (ix) in the figure show the
following structures (1) to (9), respectively. 1
[0746] GlcNAc, Gal, Man, Fuc and PA indicate N-acetylglucosamine,
galactose, mannose, fucose and a pyridylamino group, respectively.
In FIG. 4, the ratio of a sugar chain in which 1-position of fucose
was not bound to 6-position of N-acetylglucosaminethe in the
reducing end through .alpha.-bond was calculated from the area
occupied by the peaks (i) to (iv) among (i) to (ix), and the ratio
of a sugar chain in which 1-position of fucose was bound to
6-position of N-acetylglucosaminethe in the reducing end through
.alpha.-bond was calculated from the area occupied by the peaks (v)
to (ix) among (i) to (ix). Each ratio of a sugar chain was shown as
an average value of the result of two sugar chain analyses.
[0747] As a result, the ratios of a sugar chain in which 1-position
of fucose was not bound to 6-position of N-acetylglucosamine in the
reducing end through .alpha.-bond were 50%, 45% and 29% in lot 1,
lot 2 and lot 3, respectively. Herein, these samples were named
anti-GD3 chimeric antibody (50%), anti-GD3 chimeric antibody (45%)
and anti-GD3 chimeric antibody (29%).
[0748] Also, sugar chains of the anti-GD3 chimeric antibody derived
from the CHO/DG44 cell prepared in the item 2(2) of Example 1 were
analyzed in accordance with the above-described method, and it was
found that the ratio of a sugar chain in which 1-position of fucose
was not bound to 6-position of N-acetylglucosamine in the reducing
end through .alpha.-bond was 7%. Herein, the sample was named
anti-GD3 chimeric antibody (7%).
[0749] Further, the anti-GD3 chimeric antibody (45%) and anti-GD3
chimeric antibody (7%) were mixed at a ratio of anti-GD3 chimeric
antibody (45%) anti-GD3 chimeric antibody (7%)=5:3 and 1:7,
respectively. Sugar chains of the samples were analyzed in
accordance with the above-described method, and the ratios of a
sugar chain in which 1-position of fucose was not bound to
6-position of N-acetylglucosamine in the reducing end through
.alpha.-bond were 24% and 13%. Herein, they were named anti-GD3
chimeric antibody (24%) and anti-GD3 chimeric antibody (13%).
[0750] 2. Evaluation of Binding Activity to GD3 (ELISA)
[0751] The binding activities of the six kinds of the anti-GD3
chimeric antibodies having a different ratio of a sugar chain in
which 1-position of fucose was not bound to position of
N-acetylglucosamine in the reducing end through .alpha.-bond
prepared in the item 1 of Example 3 to GD3 were measured by the
ELISA shown in the item 3 of Example 1. As a result, all of the six
kinds of the anti-GD3 chimeric antibodies showed almost the same
GD3-binding activity as shown in FIG. 5, and it was found that the
ratio of a sugar chain in which 1-position of fucose was not bound
to 6-position of N-acetylglucosamine in the reducing end through
.alpha.-bond does not have influence on the antigen binding
activity of the antibody.
[0752] 3. Evaluation of ADCC Activity on Human Melanoma Cell
Line
[0753] ADCC activities of six kinds of the anti-GD3 chimeric
antibodies having a different ratio of a sugar chain in which
1-position of fucose was not bound to 6-position of
N-acetylglucosamine in the reducing end through .alpha.-bond
prepared in the item 1 of Example 3 to human melanoma cell line
G-361 (ATCC CRL1424) were measured according to the method
described in the item 2 of Example 2.
[0754] FIGS. 6 and 7 show results of the measurement of ADCC
activity of the six kinds of the anti-GD3 chimeric antibodies
having a different ratio of a sugar chain in which 1-position of
fucose was bound to 6-position of N-acetylglucosamine in the
reducing end through .alpha.-bond at various concentrations (0.0005
to 5 .mu.g/ml) using effector cells of two healthy donors (A and
B). As shown in FIGS. 6 and 7, the ADCC activity of the anti-GD3
chimeric antibodies showed a tendency to increase in proportion to
the ratio of a sugar chain in which 1-position of fucose was not
bound to 6-position of N-acetylglucosamine in the reducing end
through .alpha.-bond at each antibody concentration. The ADCC
activity decreases when the antibody concentration is low.
[0755] At an antibody concentration of 0.05 .mu.g/ml, the antibody
in which the ratio of a sugar chain in which 1-position of fucose
was not bound to 6-position of acetylglucosamine in the reducing
end through .alpha.-bond was 24%, 29%, 45% or 50% showed almost the
same high ADCC activity, but the antibody (13%) or (7%) in which
the ratio of a sugar chain in which 1-position of fucose was bound
to 6-position of N-acetylglucosamine in the reducing end through
(.alpha.-bond is less than 20%, 13% or 7% showed the low ADCC
activity. These results did not change even if the effector cell
having doner was different.
EXAMPLE 4
[0756] Activity Evaluation of Anti-CCR4 Chimeric Antibodies Having
a Different Ratio of a Sugar Chain in which 1-Position of Fucose
was not Bound to 6-Position of N-acetylglucosamine in the Reducing
End through .alpha.-Bond.
[0757] 1. Preparation of Cells Stably Producing Anti-CCR4 Chimeric
Antibody
[0758] Cells which are capable of stably producing an anti-CCR4
chimeric antibody were prepared as follows by using a tandem type
expression vector pKANTEX2160 for an anti-CCR4 chimeric antibody
described in WO01/64754.
[0759] (1) Preparation of Producing Cell Using Rat Myeloma YB2/0
Cell
[0760] After introducing 10 .mu.g of the anti-CCR4 chimeric
antibody expression vector pKANTEX2160 into 4.times.10.sup.6 cells
of rat myeloma YB2/0 cell (ATCC CRL 1662) by electroporation
[Cytotechnology, 3, 133 (1990)], the cells were suspended in 40 ml
of Hybridoma-SFM-FBS(5) [Hybridoma-SFM medium (manufactured by
Invitrogen) comprising 5% FBS (manufactured by PAA Laboratories)]
and dispensed at 200 .mu.l/well into a 96 well culture plate
(manufactured by Sumitomo Bakelite). After culturing at 37-C for 24
hours in a 5% CO.sub.2 incubator, G418 was added to give a
concentration of 1 mg/ml, followed by culturing for 1 to 2 weeks.
Culture supernatant was recovered from wells in which growth of
transformants showing G418 resistance was observed by the formation
of colonies, and antigen binding activity of the anti-CCR4 chimeric
antibody in the supernatant was measured by the ELISA described in
the item 2 of Example 4.
[0761] Regarding the transformants in wells in which production of
the anti-CCR4 chimeric antibody was observed in culture
supernatants, in order to increase an amount of the antibody
production using a DHFR gene amplification system, each of them was
suspended in the Hybridoma-SFM-FBS(5) medium comprising 1 mg/ml
G418 and 50 nmol/L DHFR inhibitor MTX (manufactured by SIGMA) to
give a density of 1 to 2.times.10.sup.5 cells/ml, and the
suspension was dispensed at 1 ml into each well of a 24 well plate
(manufactured by Greiner). After culturing at 37.degree. C. for 1
to 2 weeks in a 5% CO.sub.2 incubator, transformants showing 50
nmol/L MTX resistance were induced. Antigen binding activity of the
anti-CCR4 chimeric antibody in culture supernatants in wells in
which growth of transformants was observed was measured by the
ELISA described in the item 2 of Example 4.
[0762] Regarding the transformants in wells in which production of
the anti-CCR4 chimeric antibody was observed in culture
supernatants, the MTX concentration was increased by the same
method, and a transformant capable of growing in the
Hybridoma-SFM-FBS(5) medium comprising 200 nmol/L MTX and of
producing the anti-CCR4 chimeric antibody in a large amount was
finally obtained. The obtained transformant was made into a single
cell (cloning) by limiting dilution twice, and the obtained clone
was named KM2760#58-35-16.
[0763] (2) Preparation of Producing Cell Using CHO/DG44 Cell
[0764] After introducing 4 .mu.g of the anti-CCR4 chimeric antibody
expression vector pKANTEX2160 into 1.6.times.10.sup.6 cells of
CHO/DG44 cell by electroporation [Cytotechnology, 3, 133 (1990)],
the cells were suspended in 10 ml of IMDM-dFBS(10)-HT(1) [IMDM
medium (manufactured by Invitrogen) comprising 10% dFBS
(manufactured by Invitrogen) and 1.times. concentration of MT
supplement (manufactured by Invitrogen)] and dispensed at 100
.mu.l/well into a 96 well culture plate (manufactured by Iwaki
Glass). After culturing at 37.degree. C. for 24 hours in a 5%
CO.sub.2 incubator, the medium was changed to MD-dFBS(10) (IMDM
medium comprising 10% of dialyzed FBS) followed by culturing for 1
to 2 weeks. Culture supernatant was recovered from wells in which
the growth was observed due to formation of a transformant showing
HT-independent growth, and an expression amount of the anti-CCR4
chimeric antibody in the supernatant was measured by the ELISA
described in the item 2 of Example 4.
[0765] Regarding the transformants in wells in which production of
the anti-CCR4-chimeric antibody was observed in culture
supernatants, in order to increase an amount of the antibody
production using a DHFR gene amplification system, each of them was
suspended in the IMDM-dFBS(10) medium comprising 50 nmol/L MTX to
give a density of 1 to 2.times.10.sup.5 cells/ml, and the
suspension was dispensed at 0.5 ml into each well of a 24 well
plate (manufactured by Iwaki Glass). After culturing at 37.degree.
C. for 1 to 2 weeks in a 5% CO.sub.2 incubator, transformants
showing 50 nmol/L MTX resistance were induced. Regarding the
transformants in wells in which the growth was observed, the MTX
concentration was increased to 200 nmol/L by the similar method as
above, and a transformant capable of growing in the IMDM-dFBS(10)
medium comprising 200 nmol/L MTX and of producing the anti-CCR4
chimeric antibody in a large amount was finally obtained. The
obtained transformant was named clone 5-03.
[0766] 2. Binding Activity of Antibody to CCR4 Partial Peptide
(ELISA)
[0767] Compound 1 (SEQ ID NO:25) was selected as a human CCR4
extracellular region peptide capable of reacting with the anti-CCR4
chimeric antibody. In order to use it in the activity measurement
by ELISA, a conjugate with BSA (manufactured by Nacalai Tesque) was
prepared by the following method and used as the antigen. That is,
100 ml of a DMSO solution comprising 25 mg/ml SMCC
[4-(N-maleimidomethyl)-cyclohexane- -1-carboxylic acid
N-hydroxysuccinimide ester] (manufactured by Sigma) was added
dropwise to 900 ml of a 10 .mu.g BSA-containing PBS solution under
stirring with a vortex, followed by gently stirring for 30 minutes.
To a gel filtration column such as NAP-10 column equilibrated with
25 ml of PBS, 1 ml of the reaction solution was applied and then
eluted with 1.5 ml of PBS and the resulting eluate was used as a
BSA-SMCC solution (BSA concentration was calculated based on
A.sub.280 measurement). Next, 250 ml of PBS was added to 0.5 mg of
Compound 1 and then completely dissolved by adding 250 ml of DMF,
and the BSA-SMCC solution was added thereto under vortex, followed
by gently stirring for 3 hours. The reaction solution was dialyzed
against PBS at 4.degree. C. overnight, sodium azide was added
thereto to give a final concentration of 0.05%, and the mixture was
filtered through a 0.22 mm filter to be used as a BSA-compound 1
solution.
[0768] The prepared conjugate was dispensed at 0.05 .mu.g/ml and 50
.mu.l/well into a 96 well EIA plate (manufactured by Greiner) and
incubated for adhesion at 4.degree. C. overnight. After washing
each well with PBS, 1% BSA-PBS was added thereto in 100 .mu.l/well
and allowed to react at room temperature to block the remaining
active groups. After washing each well with Tween-PBS, a culture
supernatant of a transformant was added at 50 .mu.l/well and
allowed to react at room temperature for 1 hour. After the
reaction, each well was washed with Tween-PBS, and then a
peroxidase-labeled goat anti-human IgG(.gamma.) antibody solution
(manufactured by American Qualex) diluted 6000 times with 1%
BSA-PBS as the secondary antibody solution was added at 50
.mu.l/well and allowed to react at room temperature for 1 hour.
After the reaction and subsequent washing with Tween-PBS, the ABTS
substrate solution was added at 50 .mu.l well for color
development, and 20 minutes thereafter, the reaction was stopped by
adding a 5% SDS solution at 50 .mu.l/well. Thereafter, the
absorbance at OD.sub.415 was measured. The anti-CCR4 chimeric
antibody obtained in the item 1 of Example 4 showed the binding
activity to CCR4.
[0769] 3. Purification of Anti-CCR4 Chimeric Antibody
[0770] (1) Culturing of Producing Cell Derived from YB2/0 Cell and
Purification of Antibody
[0771] The anti-CCR4 chimeric antibody-expressing transformant cell
clone KM2760#58-35-16 obtained in the item 1(1) of Example 4 was
suspended in Hybridoma-SFM (manufactured by Invitrogen) medium
comprising 200 nmol/L MTX and 5% of Daigo's GF21 (manufactured by
Wako Pure Chemical Industries) to give a density of
2.times.10.sup.5 cells/ml and subjected to fed-batch shaking
culturing with a spinner bottle (manufactured by Iwaki Glass) in a
constant temperature chamber of 37.degree. C. After culturing for 8
to 10 days and recovering the culture supernatant, the anti-CCR4
chimeric antibody was purified using Prosep-A (manufactured by
Millipore) column and gel filtration. The purified anti-CCR4
chimeric antibody was named KM2760-1.
[0772] (2) Culturing of Producing Cell Derived from CHO-DG44 Cell
and Purification of Antibody
[0773] The anti-CCR4 chimeric antibody-producing transformant clone
5-03 obtained in the item 1(2) of Example 4 was cultured at
37.degree. C. in a 5% CO.sub.2 incubator using IMDM-dFBS(10) medium
in a 182 cm.sup.2 flask (manufactured by Greiner). When the cell
density reached confluent after several days, the culture
supernatant was discarded, and the cells were washed with 25 ml of
PBS buffer and then mixed with 35 ml of EXCELL 301 medium
(manufactured by JRH). After culturing at 37.degree. C. for 7 days
in a 5% CO.sub.2 incubator, the culture supernatant was recovered.
The anti-CCR4 chimeric, antibody was purified from the culture
supernatant using Prosep-A (manufactured by Millipore) column in
accordance with the manufacture's instructions. The purified
anti-CCR4 chimeric antibody was named KM3060.
[0774] When the binding activity to CCR4 of KM2760-t 1 and KM3060
was measured by the ELISA described in the item 2 of Example 4,
they showed equivalent binding activity.
[0775] 4. Analysis of Purified Anti-CCR4 Chimeric Antibodies
[0776] Each 4 .mu.g of the two kinds of the anti-CCR4 chimeric
antibodies produced by and purified from various animal cells,
obtained in the item 3 of Example 4 was subjected to SDS-PAGE in
accordance with a known method [Nature, 227, 680 (1970)], and the
molecular weight and purity were analyzed. In each of the purified
anti-CCR4 chimeric antibodies, a single band corresponding to the
molecular weight of about 150 Kd was found under non-reducing
conditions, and two bands of a bout 50 Kd and about 25 Kd were
found under reducing conditions. The molecular weights almost
coincided with the molecular weights deduced from the cDNA
nucleotide sequences of antibody H chain and L chain (H chain:
about 49 Kd, L chain: about 23 Kd, whole molecule about 144 Kd) and
further coincided with reports stating that an IgG type antibody
has a molecular weight of about 150 Kd under non-reducing
conditions and is degraded into H chain having a molecular weight
of about 50 Kd and L chain having a molecular weight of about 25 Kd
under reducing conditions caused by cutting an S--S bond in the
molecule (Antibodies, Chapter 14 (1988), Monoclonal Antibodies),
thus confirming that the anti-CCR4 chimeric antibody was expressed
and purified as an antibody molecule having a correct
structure.
[0777] 5. Preparation of Anti-CCR4 Chimeric Antibody Having a
Different Ratio of a Sugar Chain in which 1-Position of Fucose was
not Bound to 6-Position of N-acetylglucosamine in the Reducing End
through .alpha.-Bond
[0778] Sugar chains of the anti-CCR4 chimeric antibody KM2760-1
derived from YB2/0 cell and the anti-CCR4 chimeric antibody KM3060
derived as from CHO/DG44 cell prepared in the item 3 of Example 4
were analyzed in accordance with the method in the item 1 of
Example 3. The ratios of a sugar chain in which 1-position of
fucose was not bound to 6-position of N-acetylglucosamine in the
reducing end through .alpha.-bond were 87% and 8% in 2760-1 and
KM3060, respectively. Herein, the samples were named anti-CCR4
chimeric antibody (87%) and anti-CCR4 chimeric antibody (8%).
[0779] Furthermore, the anti-CCR4 chimeric antibody (87%) and
anti-CCR4 chimeric antibody (8%) were mixed at a ratio of anti-CCR4
chimeric antibody (87%): anti-CCR4 chimeric antibody (8%)=1:39,
16:67, 22:57, 32;47 and 42:37, respectively. Sugar chains of these
samples were analyzed in accordance with the met hod of the item 1
of Example 3. The ratios of a sugar chain in which 1-position of
fucose was not bound to 6-position of N-acetylglucosamine in the
reducing end through .alpha.-bond were 9%, 18%, 27%, 39% and 46%,
respectively. Herein, these samples were named anti-CCR4 chimeric
antibody (9%), anti-CCR4 chimeric antibody (18%), anti-CCR4
chimeric antibody (27%), anti-CCR4 chimeric antibody (39%) and
anti-CCR4 chimeric antibody (46%).
[0780] Results of the sugar chain analysis of each of the samples
are shown in FIG. 8. The ratio of a sugar chain in which 1-position
of fucose was not bound to 6-position of N-acetylglucosamine in the
reducing end through .alpha.-bond was shown as an average value of
the result of two sugar chain analyses. Hs
[0781] 6. Evaluation of Binding Activity of Antibody to CCR4
Partial Peptide (ELISA)
[0782] Binding activities of the six kinds of the anti-CCR4
chimeric antibodies having a different ratio of a sugar chain in
which 1-position of fucose was not bound to 6-position of
N-acetylglucosamine in the reducing end through .alpha.-bond
prepared in the item 5 of Example 4 to a CCR4 partial peptide were
measured in accordance with the method described in the item 2 of
Example 4.
[0783] As a result, as shown in FIG. 9, the six kinds of the
anti-CCR4 chimeric antibodies showed almost the same CCR4-binding
activity, and it was found that the ratio of a sugar chain in which
1-position of fucose was not bound to 6-position of
N-acetylglucosamine in the reducing end through .alpha.-bond does
not have influence on the antigen-binding activity of the
antibody.
[0784] 7. Evaluation of ADCC Activity on Human CCR4-High Expressing
Clone
[0785] The ADCC activity of the anti-CCR4 chimeric antibodies
against a human CCR4-high highly expressing cell was measured as
follows.
[0786] (1) Preparation of Target Cell Suspension
[0787] Cells (1.5.times.10.sup.6) of a human CCR4-highly expressing
cell, CCR4/EL-4 cell, described in WO01/64754 were prepared and a
5.55 MBq equivalent of a radioactive substance
Na.sub.2.sup.51CrO.sub.4 was added thereto, followed by reaction at
37.degree. C. for 1.5 hours to thereby label the cells with a
radioisotope. After the reaction, the cells were washed three times
by suspension in a medium and subsequent centrifugation,
resuspended in the medium and then incubated at 4.degree. C. for 30
minutes on ice for spontaneous dissociation of the radioactive
substance. After centrifugation, the cells were adjusted to give a
density of 2.times.10.sup.5 cells/ml by adding 7.5 ml of the medium
and used as a target cell suspension.
[0788] (2) Preparation of Human Effector Cell Suspension
[0789] From a healthy doner, 60 ml of peripheral blood was
collected, 0.6 ml of heparin sodium (manufactured by Shimizu
Pharmaceutical) was added thereto, followed by gently mixing. The
mixture was centrifuged (800 g, 20 minutes) to isolate a
mononuclear cell layer using Lymphoprep (manufactured by AXIS
SHIELD) in accordance with the manufacture's instructions. The
cells were washed by centrifuging (1,400 rpm, 5 minutes) three
times in a medium and then re-suspended in the medium to give a
density of 5.times.10.sup.6 cells/ml and used as a hum an effector
cell suspension.
[0790] (3) Measurement of ADCC Activity
[0791] The target cell suspension prepared in the above item (I)
was dispensed at 50 .mu.l (1.times.10.sup.4 cells/well) into each
well of a 96 well U-bottom plate (manufactured by Falcon). Next,
100 .mu.l of the effector cell suspension prepared in the above
item (2) was added thereto (5.times.10.sup.5 cells/well, ratio of
the human effector cells to the target cells was 50:1).
Furthermore, each of the anti-CCR4 chimeric antibodies was added
thereto to give a final concentration of 0.0001 to 10 .mu.g/ml
followed by reaction at 37.degree. C. for 4 hours. After the
reaction. The plate was centrifuged and the amount of .sup.51 Cr in
the supernatant was measured with a .gamma.-counter. An amount of
the spontaneously dissociated .sup.51Cr was calculated by carrying
out the same procedure as above using the medium alone instead of
the human effector cell suspension and antibody solution, and
measuring the amount of .sup.51Cr in the supernatant. An amount of
the total dissociated .sup.51Cr was calculated by carrying out the
same procedure as above using 1 mol/L hydrochloric acid solution
instead of the antibody solution and human effector cell
suspension, and measuring the amount of .sup.51Cr in the
supernatant. The ADCC activity (%) was calculated based on the
above-mentioned equation (I).
[0792] FIGS. 10 and 11 show results of the measurement of ADCC
activity of the anti-CCR4 chimeric antibodies having a different
ratio of a sugar chain in which 1-position of fucose was not bound
to 6-position of N-acetylglucosamine in the reducing end through
.alpha.-bond at various concentrations (0.001 to 10 .mu.g/ml) using
effector cells of two healthy donors (A and B), respectively. As
shown in FIGS. 10 and 11, the ADCC activity of the anti-CCR4
chimeric antibodies showed a tendency to increase in proportion to
the ratio of a sugar chain in which 1-position of fucose was not
bound to 6-position of N-acetylglucosamine in the reducing end
through .alpha.-bond at each antibody concentration. The ADCC
activity decreases when the antibody concentration is low. At an
antibody concentration of 0.01 g/ml, the antibody in which the
ratios of a sugar chain in which 1-position of fucose was not bound
to 6-position of N-acetylglucosamine in the reducing end through
.alpha.-bond was 27%, 39% and 46%, respectively, showed almost the
same high ADCC activity but the ADCC activity was low in the
antibody in which the ratio of a sugar chain in which 1-position of
fucose was not bound to 6-position of N-acetylglucosamine in the
reducing end through .alpha.-bond is less than 20%. These results
did not change even if the effector cell having doner was
different.
EXAMPLE 5
[0793] Determination of Transcription Product of
.alpha.1,6-fucosyltransfe- rase (FUT8) Gene in Host Clone:
[0794] 1. Preparation of Single-Stranded cDNA from Various
Clones
[0795] Single-stranded cDNAs were prepared from dhfr-deleted
CHO/DG44 cells and rat myeloma YB2/0 cells by the following
procedure.
[0796] The CHO/DG44 cells were suspended in IMDM medium
(manufactured by Life Technologies) supplemented with 10% FBS
(manufactured by Life Technologies) and 1.times. concentration HT
supplement (manufactured by Life Technologies), and 15 ml of the
suspension was inoculated into T75 flask for adhesion cell culture
use (manufactured by Greiner) at a density of 2.times.10.sup.5
cells/ml. Also, the YB2/0 cells were suspended in RPMI 1640 medium
(manufactured by Life Technologies) supplemented with 10% FBS
(manufactured by Life Technologies) and 4 mmol/l L-GLN
(manufactured by Life Technologies), and 15 ml of the suspension
was inoculated into T75 flask for suspension cell culture
(manufactured by Greiner) at a density of 2.times.10.sup.5;
cells/ml. They were cultured at 37.degree. C. in a 5% CO.sub.2
incubator, and 1 of respective host cells were recovered on the
1st, 2nd, 3rd, 4th and 5th days of the culturing to extract total
RNA using RNAeasy (manufactured by QIAGEN) in accordance with the
manufacture's instructions.
[0797] The total RNA was dissolved in 45 .mu.l of sterile water, 1
.mu.l of RQ1 RNase-Free DNase (manufactured by Promega), 5 .mu.l of
the attached 10.times.DNase buffer and 0.5 .mu.l of RNasin
Ribonuclease Inhibitor (manufactured by Promega) were added
thereto, followed by reaction at 37.degree. C. for 30 minutes to
degrade genomic DNA contaminated in the sample. After the reaction,
the total RNA was purified again using RNAeasy (manufactured by
QIAGEN) and dissolved in 50 .mu.l of sterile water.
[0798] In a 20 .mu.l of the reaction mixture using oligo(dT) as a
primer, single-stranded cDNA was synthesized from 3 .mu.g of each
of the obtained total RNA samples by reverse transcription reaction
using SUPERSCRIPT.TM. Preamplification System for First Strand cDNA
Synthesis (manufactured by Life Technologies) in accordance with
the manufacture's instructions. For the cloning of FUT8 and
.beta.-actin derived from respective host cells, 1.times.
concentration solution of the reaction solution was used, and for
the determination of each gene transcription amount by competitive
PCR, 50 fold-diluted aqueous solution of the reaction solution were
used. The solutions were stored at -80.degree. C. until use.
[0799] 2. Preparation Method of cDNA Partial Fragments of Chinese
Hamster FUT8 and Rat FUT8
[0800] Each cDNA partial fragment of Chinese hamster FUT8 and rat
FUT8 was prepared by the following procedure (FIG. 12).
[0801] First, primers (represented by SEQ ID NOs:4 and 5) specific
for nucleotide sequences common to human FUT8 cDNA [J. Biochem.,
121, 626 (1997)] and swine FUT8 cDNA [J. Biol. Chem., 271, 27810
(1995)] were designed.
[0802] Next, 25 .mu.l of a reaction solution [ExTaq buffer
(manufactured by Takara Shuzo), 0.2 mmol/l dNTPs and 0.5 .mu.mol/l
of the above gene-specific primers (SEQ ED NOs:4 and 5)] containing
1 .mu.l of each of the cDNA prepared from CHO cell and cDNA
prepared from YB2/0 cell, both obtained in the item 1 of Example 5
on the second day after culturing, and polymerase chain reaction
(hereinafter referred to as "PCR") was carried out by using a DNA
polymerase ExTaq (manufactured by Takara Shuzo). The PCR was
carried out by heating at 94.degree. C. for 1 minute, subsequent 30
cycles of heating at 94.degree. C. for 30 seconds, 55.degree. C.
for 30 seconds and 72.degree. C. for 2 minutes as one cycle, and
further heating at 72.degree. C. for 10 minutes.
[0803] After the PCR, the reaction solution was subjected to 0.8%
agarose gel electrophoresis, and a specific amplified fragment of
979 bp was purified using GENECLEAN Spin Kit (manufactured by BIO
101) and eluted with 10 .mu.l of sterile water (hereinafter, the
method was used for the purification of DNA fragments from agarose
gel). Into a plasmid pCR2.1, 4 .mu.l of the amplified fragment was
employed to insert in accordance with the manufacture's
instructions of TOPO TA Cloning Kit (manufactured by Invitrogen),
and E. coli XL1-Blue was transformed with the reaction solution by
the method of Cohen et al. [Proc. Natl. Acad. Sci. USA, 69, 2110
(1972)] (hereinafter, the method was used for the transformation of
E. coli). Plasmid DNA samples were isolated in accordance with a
known method [Nucleic Acids Research, 7, 1513 (1979)] (hereinafter,
the method was used for the isolation of plasmid) from
cDNA-inserted six clones among the obtained kanamycin-resistant
colonies.
[0804] The nucleotide sequences of cDNAs inserted into the plasmids
were determined by using DNA Sequencer 377 (manufactured by Parkin
Elmer) and BigDye Terminator Cycle Sequencing FS Ready Reaction kit
(manufactured by Parkin Elmer) in accordance with the method of the
manufacture's instructions. It was confirmed that all of the
inserted cDNAs of which sequences were determined by the method
encode the open reading frame (ORF) partial sequences of Chinese
hamster FUT8 and rat FUT8 (represented by SEQ ID NOs:6 and 7).
Among these, plasmid DNA samples containing absolutely no reading
error by the PCR in the sequences were selected. Herein, these
plasmids were named CHFT8-pCR2.1 and YBFT8-pCR2.1.
[0805] 3. Preparation of Chinese Hamster .beta.-Actin and Rat
.beta.-Actin cDNA
[0806] Chinese hamster .beta.-actin and rat .beta.-actin were
prepared by the following procedure (FIG. 13).
[0807] First, a forward primer specific for a common sequence
containing kit translation initiation codon (represented by SEQ ID
NO:8) and reverse primers specific for respective sequences
containing translation termination codon (represented by SEQ ID
NOs:9 and 10) were designed from Chinese hamster .beta.-actin
genomic sequence (GenBank, U20114) and rat .beta.-actin genomic
sequence [Nucleic Acids Research, 11, 1759 (1983).
[0808] Next, 25 .mu.l of a reaction solution [1.times.
concentration KOD buffer #1 (manufactured by Toyobo), 0.2 mmol/l
dNTPs, 1 mmol/l MgCl.sub.2, 0.4 .mu.mol/l of the above
gene-specific primers (SEQ ID NOs:8 and 9, or SEQ ID NOs:8 and 10)
and 5% DMSO] containing 1 .mu.l of each of the cDNA prepared from
CHO cell and cDNA prepared from YB2/0 cell, both obtained in the
item 1 of Example 5 on the second day after culturing was prepared,
and PCR was carried out by using a DNA polymerase KOD (manufactured
by Toyobo). The PCR was carried out by heating at 94.degree. C. for
4 minutes and subsequent 25 cycles of heating at 98.degree. C. for
15 seconds, 65.degree. C. for 2 seconds and 74.degree. C. for 30
seconds as one cycle.
[0809] After the PCR, the reaction solution was subjected to 0.8%
agarose gel electrophoresis, and a specific amplified fragment of
1128 bp was purified. The DNA fragment was subjected to 5'-terminal
phosphorylation using MEGALABEL (manufactured by Takara Shuzo) in
accordance with the manufacture's instructions. The DNA fragment
was recovered from the reaction solution using an ethanol
precipitation method and dissolved in 10 .mu.l of sterile
water.
[0810] Separately, 3 .mu.g of a plasmid pBluescript II KS(+)
(manufactured by Stratagene) was dissolved in 35 .mu.l of NEBuffer
2 (manufactured by New England Biolabs), and 16 units of a
restriction enzyme EcoRV (manufactured by Takara Shuzo) were added
thereto for digestion reaction at 37.degree. C. for 3 hours. To the
reaction solution, 35 .mu.l of 1 mol/l Tris-HCl buffer (pH 8.0) and
3.5 .mu.l of E. coli C15-derived alkaline phosphatase (manufactured
by Takara Shuzo) were added thereto, followed by reaction at
65.degree. C. for 30 minutes to thereby dephophorylate the DNA
terminus. The reaction solution was extracted with
phenol/chloroform, followed by ethanol precipitation, and the
recovered DNA fragment was dissolved in 100 .mu.l of sterile
water.
[0811] Each 4 .mu.l of the amplified fragment prepared from Chinese
hamster cDNA and the amplified fragment (11192 bp) prepared from
rat cDNA was mixed with 1 .mu.l of the EcoRV-EcoRV fragment (about
3.0 Kb) prepared from plasmid pBluescript II KS(+) and 5 .mu.l of
Ligation High (manufactured by Toyobo) for ligation reaction at
16.degree. C. for 30 minutes. Using the reaction solution, E. coli
XL1-Blue was transformed, and plasmid DNA samples were isolated
respectively from the obtained ampicillin-resistant clones in
accordance with a known method.
[0812] The nucleotide sequence of each cDNA inserted into the
plasmid was determined by using DNA Sequencer 377 (manufactured by
Parkin Elmer) and BigDye Terminator Cycle Sequencing FS Ready
Reaction kit (manufactured by Parkin Elmer) in accordance with the
method of the manufacture's instructions. It was confirmed that all
of the inserted cDNAs of which sequences were determined by the
method encode the ORF full sequences of Chinese hamster
.beta.-actin or rat .beta.-actin. Among these, plasmid DNA samples
containing absolutely no reading error of bases by the PCR in the
sequences were selected. Herein, the plasmids are called CHAc-pBS
and YBAc-pBS.
[0813] 4. Preparation of FUT8 Standard and Internal Control
[0814] In order to measure a transcription amount of mRNA derived
from FUT8 gene in each cell, CHFT8-pCR2.1 or YBFT8-pCR2.1, as
plasmids in which cDNA partial fragments prepared in the item 2 of
Example 5 from Chinese hamster FUT8 or rat FUT8 were inserted into
pCR2.1, respectively, were digested with a restriction enzyme
EcoRI, and the obtained linear DNAs were used as the standards for
the preparation of a calibration curve. CHFT8d-pCR2.1 and
YBFT8d-pCR2.1, which were obtained from the CHFT8-pCR2.1 and
YBFT8-pCR2.1, by deleting 203 bp between ScaI and HindIII, an inner
nucleotide sequence of Chinese hamster FUT8 and rat FUT8,
respectively, were digested with a restriction enzyme EcoRI, and
the obtained linear DNAs were used as the internal standards for
FUT8 amount determination. Details thereof are described below.
[0815] Chinese hamster FUT8 and rat FUT8 standards were prepared as
follows. In 40 .mu.l of NEBuffer 2 (manufactured by New England
Biolabs), 2 .mu.g of the plasmid CHFT8-pCR2.1 was dissolved, 24
units of a restriction enzyme EcoRI (manufactured by Takara Shuzo)
were added thereto, followed by digestion reaction at 37.degree. C.
for 3 hours. Separately, 2 .mu.g of the plasmid YBFT8-pCR2.1 was
dissolved in 40 .mu.l of NEBuffer 2 (manufactured by New England
Biolabs), and 24 units of a restriction enzyme EcoRI (manufactured
by Takara Shuzo) were added thereto, followed by digestion reaction
at 37.degree. C. for 3 hours. By subjecting a part of each of the
reaction solutions to 0.8% agarose gel electrophoresis, it was
confirmed that an EcoRI-EcoRI fragment (about 1 Kb) containing each
of cDNA partial fragments of Chinese hamster FUT8 and rat FUT8 was
separated from the plasmids CH8-pCR2.1 and YBFT8-pCR2.1 by the
above restriction enzyme digestion reactions. Each of the reaction
solutions was diluted with 1 .mu.g/ml of baker's yeast t-RNA
(manufactured by SIGMA) to give a concentration of 0.02 fg/.mu.l,
0.2 fg/.mu.l, 1 fg/.mu.l, 2 fg/.mu.l, 10 fg/.mu.l, 20 fg/.mu.l and
100 fg/.mu.l and used as the Chinese hamster FUT8 and rat FUT8
standards.
[0816] Controls of Chinese hamster FUT8 and rat FUT8 were prepared
as follows (FIG. 14). By using a DNA polymerase KOD (manufactured
by Toyobo), 25 .mu.l of a reaction solution [1.times. concentration
KOD buffer #1 (manufactured by Toyobo), 0.2 mmol/l dNTPs, 1 mmol/l
MgCl.sub.2, 0.4 .mu.mol/l gene-specific primers (SEQ ID NOs:11 and
12) and 5% DMSO] containing 5 ng of CHFT-pCR2.1 or YBFT8-pCR2.1 was
prepared, and PCR was carried out. The PCR was carried out by
heating at 94.degree. C. for 4 minutes and subsequent 25 cycles of
heating at 98.degree. C. for 1 seconds, 65.degree. C. for 2 seconds
and 74.degree. C. for 30 seconds as one cycle. After the PCR, the
reaction solution was subjected to 0.8% agarose gel
electrophoresis, and a specific amplified fragment of about 4.7 Kb
was purified. The DNA fragment was subjected to 5'-terminal
phosphorylation using MEGALABEL (manufactured by Takara Shuzo) in
accordance with the manufacture's instructions, and then the DNA
fragment was recovered from the reaction solution by ethanol
precipitation and dissolved in 50 .mu.l of sterile water. The above
obtained DNA fragment (5 .mu.l, about 4.7 kb) and 5 .mu.l of
Ligation High (manufactured by Toyobo) were mixed, followed by
self-cyclization reaction at 16.degree. C. for 30 minutes.
[0817] Using the reaction solution, E. coli DH5.alpha. was
transformed, and plasmid DNA samples were isolated in accordance
with a known method from the obtained ampicillin-resistant clones.
The nucleotide sequence of each plasmid DNA was determined by using
DNA Sequencer 377 (manufactured by Parkin Elmer) and BigDye
Terminator Cycle Sequencing FS Ready Reaction kit (manufactured by
Parkin Elmer), and it was confirmed that a 203 bp inner nucleotide
sequence between ScaI and HindIII of Chinese hamster FUT8 or rat
FUT8 was deleted. The obtained plasmids were named CHFT8d-pCR2.1 or
YBFT8d-pCR2.1, respectively.
[0818] Next, 2 .mu.g of the plasmid CHFT8d-pCR2.1 was dissolved in
40 .mu.l of NEBuffer 2 (manufactured by New England Biolabs), and
24 units of a restriction enzyme EcoRI (manufactured by Takara
Shuzo) were added thereto, followed by digestion reaction at
37.degree. C. for 3 hours. Separately, 2 .mu.g of the plasmid
YBFT8d-pCR2.1 was dissolved in 40 .mu.l of NEBuffer 2 (manufactured
by New England Biolabs), and 24 units of a restriction enzyme EcoRI
(manufactured by Takara Shuzo) were added thereto, followed by
digestion reaction at 37.degree. C. for 3 hours. A part of each of
the reaction solutions was subjected to 0.8% agarose gel
electrophoresis, and it was confirmed that an EcoRI-EcoRI fragment
(about 800 bp) containing a fragment from which 203 bp of the inner
nucleotide sequences of Chinese hamster FUT8 or rat FUT8 partial
fragments was deleted was separated from the plasmids CHFT8d-pCR2.1
or YBFT8d-pCR2.1 by the above restriction enzyme digestion
reactions. Dilutions of 2 fg/.mu.l were prepared from the reaction
solutions using 1 .mu.g/ml baker's yeast t-RNA (manufactured by
SIGMA) and used as the Chinese hamster FUT8 or rat FUT8 internal
controls.
[0819] 5. Preparation of .beta.-Actin Standard and Internal
Control
[0820] In order to measure a transcription amount of mRNA derived
from .beta.-actin gene in various host cells, CHAc-pBS and
YBAc-pBS, as plasmids in which the ORF full length of each cDNA of
Chinese hamster .beta.-actin and rat .beta.-actin prepared in the
item 3 of Example 5 was inserted into pBluescript II KS(+),
respectively, were digested with restriction enzymes HindIII and
PstI and restriction enzymes HindIII and KpnI, respectively, and
the digested linear DNAs were used as the standards for the
preparation of a calibration curve. CHAcd-pBS and YBAcd-pBS which
were obtained from the CHAc-pBS and YBAc-pBS by deleting 180 bp
between DraIII and DraIII of an inner nucleotide sequence of
Chinese hamster .beta.-actin and rat .beta.-actin were digested
with restriction enzymes HindIII and PstI and restriction enzymes
HindIII and KpnI, respectively, and the digested linear DNAs were
used as the internal controls for .beta.-actin amount
determination. Details thereof are described below.
[0821] Chinese hamster .beta.-actin and rat .beta.-actin standards
were prepared as follows. In 40 .mu.l of NEBuffer 2 (manufactured
by New England Biolabs), 2 .mu.g of the plasmid CHAc-pBS was
dissolved, and 25 units of a restriction enzyme HindIII
(manufactured by Takara Shuzo) and 20 units of PstI (manufactured
by Takara Shuzo) were added thereto, followed by digestion reaction
at 37.degree. C. for 3 hours. Separately, 2 .mu.g of the plasmid
YBAc-pBS was dissolved in 40 .mu.l of NEBuffer 2 (manufactured by
New En gland Bio labs), and 25 units of a restriction enzyme
HindIII (manufactured by Takara Shuzo) and 24 units of KpnI
(manufactured by Takara Shuzo) were added thereto, followed by
digestion reaction at 37.degree. C. for 3 hours. A part of each of
the reaction solutions was subjected to 0.8% agarose gel
electrophoresis, and it was confirmed that a HindIII-PstI fragment
and a HindIII-KpnI fragment (about 1.2 Kb) containing the full
length ORF of each cDNA of Chinese hamster .beta.-actin and rat
.beta.-actin were separated from the plasmids CHAc-pBS and YBAc-pBS
by the above restriction enzyme digestion reactions. Each of the
reaction solutions was diluted with 1 .mu.g/ml baker's yeast t-RNA
(manufactured by SIGMA) to give a concentration 2 pg/.mu.l, 1
pg/.mu.l, 200 fg/.mu.l, 100 fg/.mu.l and 20 fg/.mu.l and used as
the Chinese hamster .beta.-actin or rat .beta.-actin standards.
[0822] Chinese hamster .beta.-actin and rat .beta.-actin internal
controls were prepared as follows (FIG. 15). In 100 .mu.l of
NEBuffer 3 (manufactured by New England Biolabs) containing 100
ng/.mu.l of BSA (manufactured by New England Biolabs), 2 .mu.g of
CHAc-pBS was dissolved, and 10 units of a restriction enzyme DraIII
(manufactured by New England Biolabs) were added thereto, followed
by digestion reaction at 37.degree. C. for 3 hours. DNA fragments
were recovered from the reaction solution by ethanol precipitation
and the DNA termini were changed to blunt-ends using DNA Blunting
Kit (manufactured by Takara Shuzo) in accordance with the
manufacture's instructions, and then the reaction solution was
divided into two equal parts. First, to one part of the reaction
solution, 35 .mu.l of 1 mol/l Tris-HCl buffer (pH 8.0) and 3.5
.mu.l of E. coli C15-derived alkaline phosphatase (manufactured by
Takara Shuzo) were added thereto, followed by reaction at
65.degree. C. for 30 minutes for dephosphorylating the DNA termini.
The DNA fragment was recovered by carrying out dephosphorylation
treatment, phenol/chloroform extraction treatment and ethanol
precipitation treatment and then dissolved in 10 .mu.l of sterile
water. The remaining part of the reaction solution was subjected to
0.8% agarose gel electrophoresis to purify a DNA fragment of about
1.11 Kb containing the ORF partial fragment of Chinese hamster
.beta.-actin.
[0823] The dephosphorylated DraIII-DraIII fragment (0.5 .mu.l), 4.5
.mu.l of the DraIII-DraIII fragment of about 1.1 Kb and 5 .mu.l of
Ligation High (manufactured by Toyobo) were mixed, followed by
ligation reaction at 16.degree. C. for 30 minutes. Using the
reaction solution, E. coli DH5.alpha. was transformed, and plasmid
DNAs were isolated in accordance with a known method from the
obtained ampicillin-resistant colonies. The nucleotide sequence of
each plasmid DNA was determined using DNA Sequencer 377
(manufactured by Parkin Elmer) and BigDye Terminator Cycle
Sequencing FS Ready Reaction kit (manufactured by Parkin Elmer),
and it was confirmed that a Chinese hamster .beta.-actin
DraIII-DraIII 180 bp inserted into the plasmid was deleted. The
plasmid was named CHAcd-pBS.
[0824] Also, a plasmid in which rat .beta.-actin DraIII-DraIII 180
bp was deleted was prepared via same steps of CHAcd-pBS. The
plasmid was named YBAcd-pBS.
[0825] Next, 2 .mu.g of the plasmid CHAcd-pBS was dissolved in 40
.mu.l of NEBuffer 2 (manufactured by New England Biolabs), and 25
units of a restriction enzyme HindIII (manufactured by Takara
Shuzo) and 20 units of PstI (manufactured by Takara Shuzo) were
added thereto, followed by digestion reaction at 37.degree. C. for
3 hours. Separately, 2 .mu.g of the plasmid YBAcd-pBS was dissolved
in 40 .mu.l of NEBuffer 2 (manufactured by New England Biolabs),
and 25 units of a restriction enzyme HindIII (manufactured by
Takara Shuzo) and 24 units of KpnI (manufactured by Takara Shuzo)
were added thereto, followed by digestion reaction at 37.degree. C.
for 3 hours. A part of each of the reaction solutions was subjected
to 0.8% agarose gel electrophoresis, and it was confirmed that an
HindIII-PstI fragment and HindIII-KpnI fragment (about 1.0 Kb)
containing a fragment in which 180 bp of the inner nucleotide
sequence of the ORF full length of each cDNA of Chinese hamster
.beta.-actin and rat .beta.-actin was deleted were separated from
the plasmids CHAcd-pBS and YBAcd-pBS by the restriction enzyme
digestion reactions. Dilutions of 200 fg/.mu.l were prepared from
the reaction solutions using 1 .mu.g/ml baker's yeast t-RNA
(manufactured by SIGMA) and used as the Chinese hamster
.beta.-actin and rat .beta.-actin internal controls.
[0826] 6. Determination of Transcription Amount by Competitive
PCR
[0827] Competitive PCR was carried out by using the FUT8 internal
control DNA prepared in the item 4 of Example 5 and the host
cell-derived cDNA obtained in the item 1 of Example 5 as the
templates, and the determined value of the FUT8 transcription
product in the host clone was calculated from the relative value of
the amount of the amplified product derived from each template. On
the other hand, since it is considered that the .beta.-actin gene
is transcribed constantly in each cell and its transcription amount
is approximately the same between cells, transcription amount of
the .beta.-actin gene was determined as an indication of the
efficiency of synthesis reaction of cDNA derived from each host
clone. That is, the PCR was carried out by using the .beta.-actin
internal control DNA prepared in the item 5 of Example 5 and the
host cell-derived cDNA obtained in the item 1 of Example 5 as the
templates, the determined value of the .beta.-actin transcription
product in the host clone was calculated from the relative value of
the amount of the amplified product derived from each template.
Details thereof are described below.
[0828] The FUT8 transcription product was determined by the
following procedure. First, a set of sequence-specific primers
(represented by SEQ ID NOs:13 and 14) common to the inner sequences
of the ORF partial sequences of Chinese hamster FUT8 and rat FUT8
obtained in the item 2 of Example 5 were designed.
[0829] Next, PCR was carried out by using a DNA polymerase ExTaq
(manufactured by Takara Shuzo) in 20 .mu.l in total volume of a
reaction solution [1.times. concentration ExTaq buffer
(manufactured by Takara Shuzo), 0.2 mmol/l dNTPs, 0.5 .mu.mol/l of
the above gene-specific primers (SEQ ID NOs:13 and 14) and 5% DMSO]
containing 5 an of 50 fold-diluted cDNA solution prepared from each
of respective host clone in the item 1 of Example 5 and 5 .mu.l (10
fg) of the plasmid for internal control. The PCR was carried out by
heating at 94.degree. C. for 3 minutes and subsequent 32 cycles of
heating at 94.degree. C. for 1 minute, 60.degree. C. for 1 minute
and 72.degree. C. for 1 minute as one cycle.
[0830] Also, PCR was carried out in a series of reaction in which 5
.mu.l (0.1 fg, 1 fg, 5 fg, 10 fg, 50 fg, 100 fg, 500 fg or 1 pg) of
the FUT8 standard plasmid obtained in the item 4 of Example 5 was
added instead of the each host clone-derived cDNA, and used in the
preparation of a calibration curve for the FUT8 transcription
amount.
[0831] The .beta.-actin transcription product was determined by the
following procedure. First, two sets of respective gene-specific
primers common to the inner sequences of the ORF full lengths of
Chinese hamster .beta.-actin and rat .beta.-actin obtained in the
item 3 of Example 5 were designed (the former are represented by
SEQ ID NOs:15 and 16, and the latter are represented by SEQ ID
NOs:17 and 18).
[0832] Next, PCR was carried out by using a DNA polymerase ExTaq
(manufactured by Takara Shuzo) in 20 .mu.l in total volume of a
reaction solution [1.times. concentration ExTaq buffer
(manufactured by Takara Shuzo), 0.2 mmol/l dNTPs, 0.5 .mu.mol/l of
the above gene-specific primers (SEQ ID NOs:15 and 16, or SEQ ID
NOs:17 and 18) and 5% DMSO] containing 5 .mu.l of 50 fold-diluted
cDNA solution prepared from respective host clone in the item 1 of
Example 5 and 5 .mu.l (1 pg) of the plasmid for internal control.
The PCR was carried out by heating at 94.degree. C. for 3 minutes
and subsequent 17 cycles of heating at 94.degree. C. for 30
seconds, 65.degree. C. for 1 minute and 72.degree. C. for 2 minutes
as one cycle.
[0833] Also, PCR was carried out in a series of reaction in which 5
.mu.l (10 pg, 5 pg, 1 pg, 500 fg or 100 fg) of the .beta.-actin
standard plasmid obtained in the item 5 of Example 5 was added
instead of the each host clone-derived cDNA, and used in the
preparation of a calibration curve for the .beta.-actin
transcription amount.
1TABLE 1 Target Size of PCR amplification product (bp) gene Primer
set* Target Competitor FUT8 F: 5'-GTCCATGGTGATCCTGCAGTGTGG-3' 638
431 R: 5'-CACCAATGATATCTCCAGGTTCC-3' .beta.-Actin F:
5'-GATATCGCTGCGCTCGTTGTCGAC-3' 789 609 R: 5'-CAGGAAGGAAGGCTGGAAAA-
GAGC-3' (Chinese hamster) .beta.-Actin F:
5'-GATATCGCTGCGCTCGTCGTCGAC-3' 789 609 R: 5'-CAGGAAGGAAGGCTGGAAGA-
GAGC-3' (Rat) *F: forward primer, R: reverse primer
[0834] By carrying out PCR using the primer set shown in Table 1, a
DNA fragment having a size shown in the target column of Table 1
can be amplified from each gene transcription product and each
standard, and a DNA fragment having a size shown in the competitor
column of Table 1 can be amplified from each internal control.
[0835] After 7 .mu.l of each of the solutions after PCR was
subjected to 1.75% agarose gel electrophoresis, the gel was stained
by soaking it for 30 minutes in 1.times. concentration SYBR Green I
Nucleic Acid Gel Stain (manufactured by Molecular Probes). The
amount of the amplified DNA fragment was measured by calculating
luminescence intensity of each amplified DNA using a fluoro-imager
(FluorImager SI; manufactured by Molecular Dynamics).
[0836] The amount of an amplified product formed by PCR using a
standard plasmid as the template was measured by the
above-mentioned method, and a calibration curve was prepared by
plotting the measured values against the amounts of the standard
plasmid. Using the calibration curve, the amount of cDNA of a gene
of interest in each clone was calculated from the amount of the
amplified product when each expression clone-derived total cDNA was
used as the template, and the amount was defined as the mRNA
transcription amount in each clone.
[0837] The amount of the FUT8 transcription product in each host
clone when a rat FUT8 sequence was used as the standard and
internal control is shown in FIG. 16. Throughout the culturing
period, the CHO clone showed a transcription amount 10-fold or
higher than that of the YB2/0 clone. The tendency was also found
when a Chinese hamster FUT8 sequence was used as the standard and
internal control.
[0838] Also, the FUT8 transcription amounts are shown in Table 2 as
relative values to the amount of the .beta.-actin transcription
product. Throughout the culturing period, the FUT8 transcription
amount in the YB 2/0 clone was around 0.1% of .beta.-actin while it
was 0.5% to 2% in the CHO/DG44 clone.
[0839] The results shows that the amount of the FUT8 transcription
product in YB2/0 clone was significantly smaller than that in the
CHO/DG44 clone.
2 TABLE 2 Culture days Clone 1st 2nd 3rd 4th 5th CHO 1.95 0.90 0.57
0.52 0.54 YB2/0 0.12 0.11 0.14 0.08 0.07
EXAMPLE 6
[0840] Determination of Transcription Product of FUT8 Gene in
Anti-GD3 Chimeric Antibody-Producing Clone:
[0841] 1. Preparation of Single-Stranded cDNA Derived from Various
Antibody-Producing Clones
[0842] Single-stranded cDNA was prepared from anti-GD3 chimeric
antibody-producing cell clones DCHI01-20 and 61-33 as follows. The
clone DCHI01-20 is a transformant clone derived from the CHO/DG4
cell described in item 2(2) of Example 1. Also, the clone 61-33 is
a clone obtained by carrying out serum-free adaptation of
YB2/0-derived transformant cell clone 7-9-51 (FERM BP-6691,
International Patent Organism Depositary, National Institute of
Advanced Industrial Science and Technology) and then carrying out
single cell isolation by two limiting dilution.
[0843] The clone DCHI01-2 were suspended in EXCELL 302 medium
(manufactured by JRH BIOSCIENCES) supplemented with 3 mmol/l L-GLN
(manufactured by Life Technologies), 0.3% PLURONIC F-68
(manufactured by Life Technologies) and 0.5% fatty acid concentrate
(manufactured by Life Technologies), and 15 ml of the suspension
was inoculated into T75 flask for suspension cell culture
(manufactured by Greiner) at a density of 2.times.10.sup.5
cells/ml. Also, cells of the clone 61-33 were suspended in
Hybridoma-SFM medium (manufactured by Life Technologies)
supplemented with 0.2% BSA, and 15 ml of the suspension was
inoculated into T75 flask for suspension cell culture (manufactured
by Greiner) at a density of 2.times.10.sup.5 cells/ml. They were
cultured at 37.degree. C. in a 5% Cl % incubator, and 1, 2, 3, 4
and 5 days after culturing 1.times.10.sup.7 of respective host
cells were recovered to extract total RNA using RNAeasy
(manufactured by QIAGEN) in accordance with the manufacture's
instructions.
[0844] The total RNA was dissolved in 45 .mu.l of sterile water,
and 1 .mu.l of RQ1 RNase-Free DNase (manufactured by Promega), 5
.mu.l of the attached 10.times.DNase buffer and 0.5 .mu.l of RNasin
Ribonuclease Inhibitor (manufactured by Promega) were added
thereto, followed by reaction at 37.degree. C. for 30 minutes to
degrade genomic DNA contaminated in the sample. After the reaction,
the total RNA was purified again using RNAeasy (manufactured by
QIAGEN) and dissolved in 50 .mu.l of sterile water.
[0845] In a 20 .mu.l reaction mixture using oligo(dT) as a primer,
single-stranded cDNA was synthesized from 3 .mu.g of each of the
obtained total RNA samples by reverse transcription reaction using
SUPERSCRIPT.TM. Preamplification System for First Strand cDNA
Synthesis (manufactured by Life Technologies) in accordance with
the manufacture's instructions. The reaction solution was diluted
50-fold with water and stored at -80.degree. C. until use.
[0846] 2. Determination of Transcription Amounts of Each Gene by
Competitive PCR
[0847] The transcription amount of each of the genes of the cDNA
derived from the antibody-producing clone obtained in the item 1 of
Example 6 was determined by competitive PCR in accordance with the
item 6 of Example 5.
[0848] The FUT8 gene-derived mRNA transcription amount in each
antibody-producing clone was determined by the following
procedure.
[0849] CHFT8-pCR2.1 and YBFT8-pCR2.1, as plasmids in which cDNA
partial fragments prepared in item 2 of Ex ample 5 from Chinese
hamster FUT8 and rat FUT8, respectively, were inserted into pCR2.1,
were digested with a restriction enzyme EcoRI, and the obtained
linear DNAs were used as the standards in the preparation of a
calibration curve for determining the FUT8 transcription
amount.
[0850] CHFT8d-pCR2.1 and YBFT8d-pCR2.1, which were obtained by
deleting 203 bp between ScaI and HindIII of an inner nucleotide
sequence of Chinese hamster FUT8 and rat FUT8, respectively, in the
item 4 of Example 9 were digested with a restriction enzyme EcoRI,
and the obtained linear DNAs were used as the internal controls for
FUT8 amount determination.
[0851] PCR was carried out by using a DNA polymerase ExTaq
(manufactured by Takara Shuzo) in 20 .mu.l in total volume of a
reaction solution [1.times. concentration ExTaq buffer
(manufactured by Takara Shuzo), 0.2 mmol/l dNTPs, 0.5 .mu.mol/l
FUT8 gene-specific primers (SEQ ID NOs:13 and 14) and 5% DMSO]
containing 5 .mu.l of 50 fold-diluted cDNA solution derived from
each of the antibody-producing clone in the item 1 of Example 6 and
5 .mu.l (10 fg) of the plasmid for internal control. The PCR was
carried out by heating at 94.degree. C. for 3 minutes and
subsequent 32 cycles of heating at 94.degree. C. for 1 minute,
60.degree. C. for 1 minute and 72.degree. C. for 1 minute as one
cycle.
[0852] Also, PCR was carried out in a series of reaction in which 5
.mu.l (0.1 fg, 1 fg, 5 fg, 10 fg, 50 fg, 100 fg, 500 fg or 1 pg) of
the FUT8 standard plasmid was added instead of the each
antibody-producing clone-derived cDNA, and used in the preparation
of a calibration curve for the FUT8 transcription amount. In this
case, 1 .mu.g/ml of a baker's yeast t-RNA (manufactured by SIGMA)
was used for the dilution of the standard plasmid.
[0853] On the other hand, since it is considered that the
.beta.-actin gene is transcribed constantly in each cell and its
transcription amount is approximately the same between cells, the
transcription amount of the .beta.-actin gene was determined as an
index of the efficiency of synthesis reaction of cDNA in each
antibody-producing clone.
[0854] CHAc-pBS and YBAc-pBS as plasmids in which the OR full
length of each cDNA of Chinese hamster .beta.-actin and rat
.beta.-actin prepared in the item 3 of Example 5 were inserted into
pBluescript II KS(+), respectively, were digested with restriction
enzymes HindIII and KpnI, and the obtained linear DNAs were used as
the standards in the preparation of a calibration curve for
determining the .beta.-actin gene transcription amount.
[0855] CHAcd-pBS and YBAcd-pBS which were obtained by deleting 180
bp between DraIII and DraIII of an inner nucleotide sequence of
Chinese hamster .beta.-actin and rat .beta.-actin, respectively in
the item 5 of Example 5, were digested with restriction enzymes
HindIII and KpnI, and the obtained linear DNAs were used as the
internal controls for .beta.-actin determination.
[0856] PCR was carried out by using a DNA polymerase ExTaq
(manufactured by Takara Shuzo) in 20 .mu.l in total volume of a
reaction solution [1.times. concentration ExTaq buffer
(manufactured by Takara Shuzo), 0.2 mmol/l dNTPs, 0.5 .mu.mol/l
.beta.-actin-specific primers (SEQ ID NOs:17 and 118) and 5% DMSO]
containing 5 .mu.l of 50 fold-diluted cDNA solution derived from
each of the antibody-producing clones and 5 .mu.l (1 pg) of the
plasmid for internal control. The PCR was carried out by heating at
94.degree. C. for 3 minutes and subsequent 17 cycles of heating at
94.degree. C. for 30 seconds, 65.degree. C. for 1 minute and
72.degree. C. for 2 minutes as one cycle. Also, PCR was carried out
in a series of reaction in which 10 pg, 5 pg, 1 pg, 500 fg or 100
fg of the .beta.-actin standard plasmid was added instead of the
each antibody-producing clone-derived cDNA, and used in the
preparation of a calibration curve for the .beta.-actin
transcription amount. In this case, 1 .mu.g/ml of a baker's yeast
t-RNA (manufactured by SIGMA) was used for the dilution of standard
plasmid.
[0857] By PCR using the primer set described in Table 1, a DNA
fragment having a size shown in the target column of Table 1 can be
amplified from each gene transcription product and each standard,
and a DNA fragment having a size shown in the competitor column of
Table 1 can be amplified from each internal control.
[0858] After 7 .mu.l of the solutions after PCR was subjected to
1.75% agarose gel electrophoresis, the gel was stained by soaking
it for 30 minutes in 1.times. concentration SYBR Green I Nucleic
Acid Gel Stain (manufactured by Molecular Probes). The amount of
the amplified DNA fragment was measured by calculating luminescence
intensity of each amplified DNA using a fluoro-imager (FluorImager
SI; manufactured by Molecular Dynamics).
[0859] The amount of the amplified product formed by PCR which used
a standard plasmid as the template was measured by the above
method, and a calibration curve was pre pared by plotting the
measured values against the amounts of the standard plasmid. Using
the calibration curve, the amount of cDNA of a gene of interest in
each clone was calculated from the amount of the amplified product
when each antibody-producing clone-derived total cDNA was used as
the template, and the value was defined as the mRNA transcription
amount in each clone.
[0860] The FUT8 transcription amounts are shown in Table 3 as
relative values to the amount of the .beta.-actin transcription
product. Throughout the culturing period, the FUT8 transcription
amount in the YB2/0 cell-derived antibody-producing clone 61-33 was
0.3% or less of .beta.-actin while the FUT8 transcription amount in
the CHO-derived antibody producing clone DCHI01-20 was 0.7% to 1.5%
in the CHO cell-derived antibody-producing cell. The results shows
that the amount of the FUT8 transcription product in the YB2/0
cell-derived antibody-producing clone was significantly less than
that in the CHO cell-derived antibody-producing clone.
3 TABLE 3 Culture days Clone 1st 2nd 3rd 4th 5th DCHI01-20 0.75
0.73 0.99 1.31 1.36 61-33 0.16 0.19 0.24 0.30 <0.10
EXAMPLE 7
[0861] Preparation of Lectin-Resistant CHO/DG44 Cell and Production
of Antibody Using the Cell
[0862] 1. Preparation of Lectin-Resistant CHO/DG44
[0863] CHO/DG44 cells were cultured in a 75 cm.sup.2 flask for
adhesion culture (manufactured by Greiner) in IMDM-FBS(10)-HT(1)
medium [IMDM medium comprising 10% of FBS and 1.times.
concentration of HT supplement (manufactured by GIBCO BRL)] to grow
until they reached a stage of just before confluent. After washing
the cells with 5 ml of PBS (manufactured by Invitrogen), 1.5 ml of
0.05% trypsin (manufactured by Invitrogen) diluted with Dulbecco
PBS was added thereto and cultured at 37.degree. C. for 5 minutes
to remove the cells from the flask bottom. The removed cells were
recovered by a centrifugation operation generally used in cell
culture and suspended in IMDM-FBS(10) medium to give a density of
1.times.10.sup.5 cells/ml, and then 0.1 .mu.g/ml of an alkylating
agent N-methyl-N'-nitro-N-nitrosoguanidine (hereinafter referred to
as "MNNG", manufactured by Sigma) was added or not added thereto.
After culturing at 37.degree. C. for 3 days in a CO.sub.2 incubator
(manufactured by TABAI), the culture supernatant was discarded, and
the cells were again washed, removed and recovered by the same
operations as described above, suspended in IMDM-FBS(10)-HT(1)
medium and then inoculated into an adhesion culture 96 well plate
(manufactured by IWAKI Glass) to give a density of 1.times.10.sup.3
cells/well. To each well, as the final concentration in medium, 1
mg/ml Lens culinaris agglutinin (hereinafter referred to as "LCA",
manufactured by Vector), 1 mg/ml Aleuria aurantia agglutinin
(Aleuria aurantia lectin; herein after referred to as "AAL",
manufactured by Vector) or 1 mg/ml kidney bean agglutinin
(Phaseolus vulgaris leucoagglutinin; hereinafter referred to as
"L-PHA", manufactured by Vector) was added. After culturing at
37.degree. C. for 2 weeks in a CO.sub.2 incubator, the appeared
colonies were obtained as lectin-resistant clone CHO/DG44.
Regarding the obtained lectin-resistant clone CHO/DG44, an
LCA-resistant clone was named clone CHO-LCA, an AAL-resistant clone
was named clone CHO-AAL and an L-PHA-resistant clone was named
clone CHO-PHA. When the resistance of these clones to various kinds
of lectin was examined, it was found that the clone CHO-LCA was
also resistant to AAL and the clone CHO-AAL was also resistant LCA.
In addition, the clone CHO-LCA and the clone CHO-AAL, also showed a
resistance to a lectin which recognizes a sugar chain structure
identical to the sugar chain structure recognized by LCA and AAL,
namely a lectin which recognizes a sugar chain structure in which
6-position of fucose is bound to 1-position of N-acetylglucosamine
residue in the reducing end through .alpha.-bond in the
N-glycoside-linked sugar chain. Specifically, it was found that the
clone CHO-LCA and the clone CHO-AAL can show resistance and survive
even in a medium supplemented with 1 mg/ml at a final concentration
of a pea agglutinin (Pisum sativum agglutinin; hereinafter referred
to as "PSA", manufactured by Vector). In addition, even when the
alkylating agent MNNG was not added, it was able to obtain
lectin-resistant clones by increasing the number of cells to be
treated. Hereinafter, these clones were used in analyses.
[0864] 2. Preparation of Anti-CCR4 Chimeric Antibody-Producing
Cell
[0865] An anti-CCR4 chimeric antibody expression plasmid
pKANTEX2160 was introduced into the three kinds of the
lectin-resistant clones obtained in the item 1 of Example 7 by the
method described in Example 4, and gene amplification by a drug MTX
was carried out to prepare an anti-CCR4 human chimeric
antibody-producing clone. By measuring an amount of antibody
expression by the ELISA described in the item 2 of Example 4,
antibody-expressing transformants were obtained from each of the
clone CHO-LCA, the clone CHO-AAL and the clone CHO-PHA. Regarding
each of the obtained transformants, a transformant derived from the
clone CHO-LCA was named clone CHO/CCR4-LCA, a transformant derived
from the clone CHO-AAL was named clone CHO/CCR4-AAL and a
transformant derived from the clone CHO-PHA was named clone
CHO/CCR4-PHA. Further, the clone CHO/CCR4-LCA, as a name of
Nega-13, has been deposited on Sep. 26, 2001, as FERM BP-7756 in
International Patent Organism Depositary, National Institute of
Advanced Industrial Science and Technology (Tsukuba Central 6, 1,
Higashi 1-Chome Tsukuba-shi, Ibaraki-ken, Japan).
[0866] 3. Production of High ADCC Activity Antibody by
Lectin-Resistant CHO Cell
[0867] Using the three kinds of the transformants obtained in the
item 2 of Example 7, purified antibodies were obtained by the
method described in the item 3 of Example 4. The antigen binding
activity of the purified anti-CCR4 chimeric antibodies was
evaluated by the ELISA described in the item 2 of Example 4. The
antibodies produced by all transformants showed an antigen binding
activity similar to that of the antibody produced by a recombinant
clone (clone 5-03) prepared in Example 4 using normal CHO/DG44 cell
as the host. Using these purified antibodies, ADCC activity of each
of the anti-CCR4 chimeric antibodies was evaluated in accordance
with the method described in the item 7 of Example 4. The results
are shown in FIG. 17. In comparison with the antibody produced by
the clone 5-03, about 100 fold-increased. ADCC activity was
observed in the antibodies produced by the clone CHO/CCR4-LCA and
the clone CHO/CCR4-AAL. On the other hand, no significant increase
in the ADCC activity was observed in the antibody produced by the
clone CHO/CCR4-PHA. Also, when ADCC activities of the antibodies
produced by the clone CHO/CCR4-LCA and the YB2/0 cell-derived clone
were compared in accordance with the method described in the item 7
of Example 4, it was found that the antibody produced by the clone
CHO/CCR4-LCA shows higher ADCC activity, similar to the case of the
antibody KM2760-1 produced by the YB2/0 cell-derived clone prepared
in the item 1 of Example 4 (FIG. 18).
[0868] 4. Sugar Chain Analysis of Antibody Produced by
Lectin-Resistant CHO Cell
[0869] Sugar chains of the anti-CCR4 chimeric antibodies purified
in the item 3 of Example 7 were analyzed according to the method
described in the item 3 of Example 3. FIG. 19 shows elution
patterns of the purified PA-treated sugar chains of the various
anti-CCR4 chimeric antibodies.
[0870] Table 4 shows the result of ratios (%) of a sugar chain in
which 1-position of fucose was not bound to 6-position of
N-acetylglucosamine in the reducing end obtained by the result of
the sugar chain analysis of the anti-CCR4 chimeric antibodies
produced by various lectin-resistant clones.
4 TABLE 4 Antibody Ratio of .alpha.1,6-Fucose-free producing cell
sugar chain (%) Clone 5-03 9 Clone CHO/CCR4-LCA 48 Clone
CHO/CCR4-AAL 27 Clone CHO/CCR4-PHA 8
[0871] In comparison with the antibody produced by the clone 5-03,
the ratio of a sugar chain in which 1-position of fucose was not
bound to 6-position of N-acetylglucosamine in the reducing end
through .alpha.-bond was increased from 9% to 48% in the antibody
produced by the clone CHO/CCR4-LCA. The ratio of
.alpha.1,6-fucose-free sugar chains was increased from 9% to 27% in
the antibody produced by the clone CHO/CCR4-AAL. On the other hand,
changes in the sugar chain pattern and the ratio of
.alpha.1,6-fucose-free sugar chains were hardly found in the clone
CHO/CCR4-PHA when compared with the clone 5-03.
EXAMPLE 8
[0872] Analysis of Lectin-Resistant CHO Clone:
[0873] 1. Analysis of Expression Amount of GMD Enzyme in Anti-CCR4
Chimeric Antibody-Producing Clone CHO/CCR4-LCA
[0874] The expression amount of each of the genes of GMD, GFPP and
FX, known as fucose biosynthesis enzymes and FUT8 as a
fucosyltransferase, in the anti-CCR4 human chimeric
antibody-producing clone CHO/CCR4-LCA obtained in Example 7, was
analyzed using RT-PCR method.,
[0875] (1) Preparation of RNA from Various Clones
[0876] Each of CHO/DG44 cell, the anti-CCR4 human chimeric
antibody-producing clone 5-03 obtained in the item 1(2) of Example
4 and the anti-CCR4 chimeric antibody-producing clone CHO/CCR4-LCA
obtained in the item 2 of Example 7 was subcultured at 37.degree.
C. in a 5% CO.sub.2 incubator and then cultured for 4 days. After
culturing, RNA was prepared from 1.times.10.sup.7 cells of each
clone using RNeasy Protect Mini Kit (manufactured by QIAGEN) in
accordance with the manufacture's instructions. Subsequently,
single-stranded cDNA was synthesized from 5 .mu.g of each RNA in a
20 .mu.l of a reaction solution using SUPER SCRIPT First-Strand
Synthesis System for RT-PCR (manufactured by GIBCO BRL) in
accordance with the manufacture's instructions.
[0877] (2) Analysis of Expression Amount of GMD Gene Using
RT-PCR
[0878] In order to amplify GMD cDNA by PCR, a 24 mer synthetic DNA
primer having the nucleotide sequence shown by SEQ ID NO. 32 and a
26 mer synthetic DNA primer having the nucleotide sequence shown by
SEQ ID NO:33 were prepared based on the CHO cell-derived GMD cDNA
sequence shown in the item 1 of Reference Example 2.
[0879] Next, 20 .mu.l of a reaction solution [1.times.
concentration Ex Taq buffer (manufactured by Takara Shuzo), 0.2
mmol/L dNTPs, 0.5 unit of Ex Taq polymerase (manufactured by Takara
Shuzo) and 0.5 .mu.L of the synthetic, DNA primers of SEQ ID NOs:32
and 33] containing 0.5 .mu.l of the single-stranded cDNA derived
from each clone in the item 1(1) of Example 8 as the template was
prepared, and PCR was carried out by using DNA Thermal Cycler 480
(manufactured by Perkin Elmer) by heating at 94.degree. C. for 5
minutes and subsequent 30 cycles of heating at 94.degree. C. for 1
minute and 68.degree. C. for 2 minutes as one cycle. After
subjecting 10 .mu.l of the PCR reaction solution to agarose
electrophoresis, DNA fragments were stained with Cyber Green
(manufactured by BMA) and then the amount of the DNA fragment of
about 350 bp was measured by using Fluor Imager SI (manufactured by
Molecular Dynamics).
[0880] (3) Analysis of Expression Amount of GFPP Gene Using
RT-PCR
[0881] In order to amplify GFPP cDNA by PCR, a 27 mer synthetic DNA
primer having the nucleotide sequence shown by SEQ ID NO:34 and a
23 mer synthetic DNA primer having the nucleotide sequence shown by
SEQ ID NO:35 were prepared based on the CHO cell-derived GFPP cDNA
sequence obtained in the item 2 of Reference Example 1.
[0882] Next, 20 .mu.l of a reaction solution [1.times.Ex Taq buffer
(manufactured by Takara Shuzo), 0.2 mmol/L dNTPs, 0.5 unit of Ex
Taq polymerase (manufactured by Takara Shuzo) and 0.5 .mu.l mol/L
of the synthetic DNA primers of SEQ ID NOs:34 and 35] containing
0.5 .mu.l of the single-stranded cDNA prepared from each clone in
the item 1(1) of Example 8 as the template was prepared, and PCR
was carried out by using DNA Thermal Cycler 480 (manufactured by
Perkin Elmer) by heating at 94.degree. C. for 5 minutes and
subsequent 24 cycles of heating at 94.degree. C. for 1 minute and
68.degree. C. for 2 minutes as one cycle. After subjecting 10 .mu.l
of the PCR reaction solution to agarose electrophoresis, DNA
fragments were stained with Cyber Green (manufactured by BMA) and
then the amount of the DNA fragment of about 600 bp was measured by
using Fluor Imager SI (manufactured by Molecular Dynamics).
[0883] (4) Analysis of Expression Amount of FX Gene Using
RT-PCR
[0884] In order to amplify FX cDNA by PCR, a 28 mer synthetic DNA
primer having the nucleotide sequence shown by SEQ ID NO:36 and a
28 mer synthetic DNA primer having the nucleotide sequence shown by
SEQ ID NO:37 were prepared based on the CHO cell-derived FX cDNA
sequence shown in the item 1 of Reference Example 1.
[0885] Next, 20 .mu.l of a reaction solution [1.times.
concentration Ex Taq buffer (manufactured by Takara Shuzo), 0.2
mmol/L dNTPs, 0.5 unit of Ex Taq polymerase (manufactured by Takara
Shuzo) and 0.5 .mu.mol/L of the synthetic DNA primers of SEQ ID
NO:36 and SEQ ID NO:7] containing 0.5 .mu.l of the single-stranded
cDNA derived from each clone in the item 1(1) of Example 8 as the
template was prepared, and PCR was carried out by using DNA Thermal
Cycler 480 (manufactured by Perkin Elmer) by heating at 94.degree.
C. for 5 minutes and subsequent 22 cycles of heating at 94.degree.
C. for 1 minute and 68.degree. C. for 2 minutes as one cycle. After
subjecting 10 .mu.l of the PCR reaction solution to agarose
electrophoresis, DNA fragments were stained with Cyber Green
(manufactured by BMA) and then the amount of the DNA fragment of
about 300 bp was measured by using Fluor Imager Si (manufactured by
Molecular Dynamics).
[0886] (5) Analysis of Expression Amount of FUT8 Gene Using
RT-PCR
[0887] In order to amplify FUT8 cDNA by PCR, 20 .mu.l of a reaction
solution [1.times.Ex Taq buffer (manufactured by Takara Shuzo), 0.2
mmol/L dNTPs, 0.5 unit of Ex Taq polymerase (manufactured by Takara
Shuzo) and 0.5 .mu.mol/L of the synthetic DNA primers of SEQ ID
NOs:13 and 14] containing 0.5 .mu.l of the single-stranded cDNA
derived from each clone in the item 1(1) of Example 8 as the
template was prepared, and PCR was carried out by using DNA Thermal
Cycler 480 (manufactured by Perkin Elmer) by heating at 94.degree.
C. for 5 minutes and subsequent 20 cycles of heating at 94.degree.
C. for 1 minute and 68.degree. C. for 2 minutes as one cycle. After
subjecting 10 .mu.l of the PCR reaction solution to agarose
electrophoresis, DNA fragments were stained with Cyber Green
(manufactured by BMA) and then amount of the DNA fragment of about
600 bp was measured using Fluor Imager SI (manufactured by
Molecular Dynamics).
[0888] (6) Analysis of Expression Amount of .beta.-Actin Gene Using
RT-PCR
[0889] In order to amplify .beta.-actin cDNA by PCR, 20 .mu.l of a
reaction solution [1.times.Ex Taq buffer (manufactured by Takara
Shuzo), 0.2 mmol/L dNTPs, 0.5 unit of Ex Taq polymerase
(manufactured by Takara Shuzo) and 0.5 .mu.mol/L of the synthetic
DNA primers of SEQ ID NOs:15 and 16] containing 0.5 .mu.l of the
single-stranded cDNA derived from each clone in the item 1(1) of
Example 8 as the template was prepared, and PCR was carried out by
using DNA Thermal Cycler 480 (manufactured by Perkin Elmer) by
heating at 94.degree. C. for 5 minutes and subsequent 14 cycles of
heating at 94.degree. C. for 1 minute and 68.degree. C. for 2
minutes as one cycle. After subjecting 10 .mu.l of the PCR reaction
solution to agarose electrophoresis, DNA fragments were stained
with Cyber Green (manufactured by BMA) and then the amount of the
DNA fragment of about 800 bp was measured using Fluor Imager SI
(manufactured by Molecular Dynamics).
[0890] (7) Expression Amount of GMD, GFPP, FX and FUT8 Genes in
Each Clone
[0891] The amount of the PCR-amplified fragment of each gene in the
clone 5-03 and the clone CHO/CCR4-LCA was calculated by dividing
values of the amounts of PCR-amplified fragments derived from GMD,
GFPP, FX and FUT cDNA in each clone measured in the items 1(2) to
1(6) of Example 8 by the value of the amount of PCR-amplified
fragment derived from .beta.-actin cDNA in each clone, and defining
the amount of the PCR-amplified fragments in CHO/DG44 cell as 1.
The results are shown in
5 TABLE 5 GMD GEPP FX FUT8 Clone CHO/DG44 1 1 1 1 Clone CHO/DG44
1.107 0.793 1.093 0.901 Anti-CCR4 antibody-producing cell Clone
5-03 Derived from clone 5-03 0.160 0.886 0.920 0.875 LCA-resistant
cell CHO/CCR4-LCA
[0892] As shown in Table 5, the expression amount of GMD gene in
the clone CHO/CCR4-LCA was decreased to about {fraction (1/10)} in
comparison with other clones. In this case, the test was
independently carried out twice, and the average value was
used.
[0893] 2. Analysis Using Anti-CCR4 Chimeric Antibody-Producing
Clone CHO/CCR4-LCA in which GMD Gene was Forced to Express
[0894] (1) Construction of CHO Cell-Derived GMD Gene Expression
Vector pAGE249GMD
[0895] Based on the CHO cell-derived GMD cDNA sequence obtained in
the item 1 of Reference Example 2, a 28 mer primer having the
nucleotide sequence shown by SEQ ID: NO:38 and a 29 mer primer
having the nucleotide sequence shown by SEQ ID NO:39 were prepared.
Next, 20 .mu.l of a reaction solution [1.times. concentration Ex
Taq buffer (manufactured by Takara Shuzo), 0.2 mmol/L dNTPs, 0.5
unit of Ex Taq polymerase (manufactured by Takara Shuzo) and 0.5
.mu.mol/L of the synthetic DNA primers of SEQ ID NOs:38 and 391
containing 0.5 .mu.l of the CHO cell-derived single-stranded cDNA
prepared in the item 1(1) of Example 8 as the template was
prepared, and PCR was carried out by using DNA Thermal Cycler 480
(manufactured by Perkin Elmer) by heating at 94.degree. C. for 5
minutes and subsequently 8 cycles of heating at 94.degree. C. for 1
minute, 58.degree. C. for 1 minute and 72.degree. C. for 1 minute
as one cycle, and then 22 cycles of heating at 94.degree. C. for 1
minute and 68.degree. C. as one cycle. After completion of the
reaction, the PCR reaction solution was fractionated by agarose
electrophoresis, and then a DNA fragment of about 600 bp was
recovered. The recovered DNA fragment was connected to pT7Blue(R)
vector (manufactured by Novagen) by using DNA Ligation Kit
(manufactured by Takara Shuzo), and E. coli DH5.alpha.
(manufactured by Toyobo) was transformed using the obtained
recombinant plasmid DNA to obtain a plasmid mt-C (FIG. 20).
[0896] Next, based on the CHO cell-derived GMD cDNA sequence
obtained in the item 1 of Reference Example 2, a 45 mer primer
having the nucleotide sequence shown by SEQ ID NO:40 and a 31 mer
primer having the nucleotide sequence shown by SEQ ID NO:41 were
prepared. Next, 20 .mu.l of a reaction solution [1.times.Ex Taq
buffer (manufactured by Takara Shuzo), 0.2 mmol/L dNTPs, 0.5 unit
of Ex Taq polymerase (manufactured by Takara Shuzo) and 0.5
.mu.mol/L of the synthetic DNA primers of SEQ ID NOs:40 and 41]
containing 0.5 .mu.l of the CHO cell-derived single-stranded cDNA
prepared in the item 1(1) of Example 8 as the template was
prepared, and PCR was carried out by using DNA Thermal Cycler 480
(manufactured by Perkin Elmer) by heating at 94.degree. C. for 5
minutes and subsequently 8 cycles of heating at 94.degree. C. for 1
minute, 57.degree. C. for 1 minute and 72.degree. C. for 1 minute
as one cycle, and then 22 cycles of heating at 94.degree. C. for 1
minute and 68.degree. C. for 2 minutes as one cycle. After
completion of the reaction, the PCR reaction solution was
fractionated by agarose electrophoresis, and then a DNA fragment of
about 150 bp was recovered. The recovered DNA fragment was
connected to pT7Blue(R) vector (manufactured by Novagen) by using
DNA Ligation Kit (manufactured by Takara Shuzo), and E. coli
DH5.alpha. (manufactured by Toyobo) was transformed using the
obtained recombinant plasmid DNA to obtain a plasmid ATG (FIG.
21).
[0897] Next, 3 .mu.g of the plasmid CHO-GMD prepared in the item 1
of Reference Example 2 was allowed to react with a restriction
enzyme SacI (manufactured by Takara Shuzo) at 37.degree. C. for 16
hours, a DNA was recovered by carrying, out phenol/chloroform
extraction and ethanol precipitation and allowed to react with a
restriction enzyme EcoRI (manufactured by Takara Shuzo) at
37.degree. C. for 16 hours. A digest DNA was fractionated by
agarose electrophoresis and then a DNA fragment of about 900 bp was
recovered. The plasmid mt-C (1.4 .mu.g) was allowed to react with a
restriction enzyme SacI (manufactured by Takara Shuzo) at
37.degree. C. for 16 hours, DNA was recovered by carrying out
phenol/chloroform extraction and ethanol precipitation and allowed
to react with a restriction enzyme EcoRI (manufactured by Takara
Shuzo) at 37.degree. C. for 16 hours. A digested DNA was
fractionated by agarose electrophoresis and, then a DNA fragment of
about 3.1 kbp was recovered. The recovered DNA fragments were
ligated by using DNA Ligation Kit (manufactured by Takara Shuzo),
and E. coli DH5.alpha. was transformed using the obtained
recombinant plasmid DNA to obtain a plasmid WT-N(-) (FIG. 22).
[0898] Next, 2 .mu.g of the plasmid WT-N(-) was allowed to react
with a restriction enzyme BamHI (manufactured by Takara Shuzo) at
37.degree. C. for 16 hours, DNA was recovered by carrying out
phenol/chloroform extraction and ethanol precipitation and allowed
to react with a restriction enzyme EcoRI (manufactured by Takara
Shuzo) at 37.degree. C. for 16 hours. A digested DNA was
fractionated by agarose electrophoresis and then a DNA fragment of
about 1 kbp was recovered by using Gene Clean II Kit (manufactured
by BIO 101) in accordance with the manufacture's instructions. The
plasmid pBluescript SK(-) (3 .mu.g; manufactured by Stratagene) was
allowed to react with a restriction enzyme BamHI (manufactured by
Takara Shuzo) at 37.degree. C. for 16 hours, DNA was recovered by
carrying out phenol/chloroform extraction and ethanol precipitation
and allowed to react with a restriction enzyme EcoRI (manufactured
by Takara Shuzo) at 37.degree. C. for 16 hours. A digested DNA was
fractionated by agarose electrophoresis and then a DNA fragment of
about 3 kbp was recovered. The respective recovered DNA fragments
were ligated by using DNA Ligation Kit (manufactured by Takara
Shuzo), and E. coli DH5.alpha. was trans formed using the obtained
recombinant plasmid DNA to obtain a plasmid WT-N(-) in pBS (cf.
FIG. 23).
[0899] Next, 2 .mu.g of the plasmid WT-N(-) in pBS was allowed to
react with a restriction enzyme HindIII (manufactured by Takara
Shuzo) at 37.degree. C. for 16 hours, DNA was recovered by carrying
out phenol/chloroform extraction and ethanol precipitation and
allowed to react with a restriction enzyme EcoRI (manufactured by
Takara Shuzo) at 37.degree. C. for 16 hours. A digested DNA was
fractionated by agarose electrophoresis and then a DNA fragment of
about 4 kbp was recovered. After 2 .mu.g of the plasmid ATG was
allowed to react with a restriction enzyme HindIII (manufactured by
Takara Shuzo) at 37.degree. C. for 16 hours, DNA was recovered by
carrying out phenol/chloroform extraction and ethanol precipitation
and allowed to react with a restriction enzyme EcoRI (manufactured
by Takara Shuzo) at 37.degree. C. for 16 hours. A digested DNA was
fractionated by agarose electrophoresis and then a DNA fragment of
about 150 bp was recovered. The respective recovered DNA fragments
were ligated by using DNA Ligation Kit (manufactured by Takara
Shuzo), and E. coli DH5.alpha. was transformed using the obtained
recombinant plasmid DNA to obtain a plasmid WT in pBS (FIG.
24).
[0900] Next, 2 .mu.g of the plasmid pAGE249 was allowed to react
with restriction enzymes HindIII and BamHI (both manufactured by
Takara Shuzo) at 37.degree. C. for 16 hours. A digested DNA was
fractionated by agarose electrophoresis and then a DNA fragment of
about 6.5 kbp was recovered. The plasmid WT (2 .mu.g) in pBS was
allowed to react with restriction enzymes HindIII and BamHI (both
manufactured by Takara Shuzo) at 37.degree. C. for 16 hours. A
digested DNA was fractionated by agarose electrophoresis and then a
DNA fragment of about 1.2 kbp was recovered. The respective
recovered DNA fragments were ligated by using DNA Ligation Kit
(manufactured by Takara Shuzo), and E. coli DH5.alpha. was
transform ed using the obtained recombinant plasmid DNA to obtain a
plasmid pAGE249GM (FIG. 25).
[0901] (2) Stable Expression of GMD Gene in Clone CHO/CCR4-LCA
[0902] The CHO cell-derived GMD gene expression vector pAGE249GMD
(5 .mu.g) which was made into linear form by digesting it with a
restriction enzyme FspI (manufactured by NEW ENGLAND BIOLABS), was
introduced into at 1.6.times.10.sup.6 cells of the clone
CHO/CCR4-LCA by electroporation [Cytotechnology 3, 133 (1990)]. Th
en, the cells were suspended in 30 ml of IMDM-dFBS(10) medium (M
medium (manufactured by GIBCO BRL) supplemented with 10% of dFBS]
comprising 200 nmol/L MTX (manufactured by SIGMA), and cultured in
a 182 cm.sup.2 flask (manufactured by Greiner) at 37.degree. C. for
24 hours in a 5% CO.sub.2 incubator. After culturing, the medium
was changed to IMDM-dFBS(10) medium containing 0.5 mg/ml hygromycin
and 200 nmol/L MTX (manufactured by SIGMA), followed by culturing
for 19 days to obtain colonies of hygromycin-resistant
transformants.
[0903] In the same manner, the pAGE249 vector was introduced into
the clone CHO/CCR4-LCA by the same method as above to obtain
colonies of hygromycin-resistant transformants.
[0904] (3) Culturing of GMD Gene-Expressed Clone CHO/CCR4-LCA and
Purification of Antibody
[0905] Using IMDM-dFBS(10) medium comprising 200 nmol/L MTX
(manufactured by SIGMA) and 0.5 mg/ml hygromycin, the
GMD-expressing transformant cells obtained in the item 2(2) of
Example 8 were cultured in a 182 cm.sup.2 flask (manufactured by
Greiner) at 37.degree. C. in a 5% CO.sub.2 incubator. Several days
thereafter, when the cell density reached confluent, the culture
supernatant was discarded and the cells were washed with 25 ml of
PBS buffer (manufactured by GIBCO BRL) and mixed with 35 ml of
EXCELL301 medium (manufactured by JRH). After culturing at
37.degree. C. in a 5% CO.sub.2 incubator for 7 days, the culture
supernatant was recovered. An anti-CCR4 chimeric antibody was
purified from the culture supernatant by using Prosep-A column
(manufactured by Millipore) in accordance with the manufacture's
instructions.
[0906] In the same manner, the pAGE249 vector-introduced
transformant cells were cultured by the same method as above and
then anti-CCR4 chimeric antibody was recovered and purified from
the culture supernatant.
[0907] (4) Measurement of Lectin Resistance in Transformed
Cells
[0908] The GMD gene-expressing transformant cells obtained in the
item 2(2) of Example 8 were suspended in IMDM-dFBS(10) medium
comprising 200 nmol/L MTX (manufactured by SIGMA) and 0.5 mg/ml
hygromycin to give a density of 6.times.10.sup.4 cells/ml, and the
suspension was dispensed at 50 l/well into a 96 well culture plate
(manufactured by Iwaki Glass). Next, a medium prepared by
suspending LCA (Lens culinaris agglutinin: manufactured by Vector
Laboratories) at concentrations of 0 mg/ml, 0.4 mg/ml, 1.6 mg/ml or
4 mg/ml in IMDM-dFBS(10) medium containing 200 nmol/L MTX
(manufactured by SIGMA) and 0.5 mg/ml hygromycin was added to the
plate at 50 .mu.l/well, followed by culturing at 37.degree. C. for
96 hours in a 5% CO.sub.2 incubator. After culturing, WST-1
(manufactured by Boehringer) was added at 10 .mu.l/well and
incubated at 37.degree. C. for 30 minutes in a 5% CO.sub.2
incubator for color development, and then the absorbance at 450 nm
and 595 nm (hereinafter referred to as "OD450" and "OD595",
respectively) was measured by using Microplate Reader (manufactured
by BIO-RAD). In the same manner, the pAGE249 vector-introduced
transformant cells were measured by the same method as above. The
above-mentioned test was carried out twice independently.
[0909] FIG. 26 shows the number of survived cells in each well by
percentage when a value calculated by subtracting OD595 from OD450
measured in the above is used as the survived number of each cell
group and the number of survived cells in each of the LCA-free
wells is defined as 100%. As shown in FIG. 26, decrease in the
LCA-resistance was observed in the GMD-expressed clone
CHO/CCR4-LCA, and the survival ratio was about 40% in the presence
of 0.2 mg/ml LCA and the survival ratio was about 20% in the
presence of 0.8 mg/ml LCA. On the other hand, in the pAGE249
vector-introduced stain CHO/CCR4-LCA, the survival ratio was 100%
in the presence of 0.2 mg/ml LCA and the survival ratio was about
80% even in the presence of 0.8 mg/ml LCA. Based on the above
results, it was suggested that expression amount of GMD gene in the
clone CHO/CCR4-LCA was decreased and, as a result, the resistance
against LCA was obtained.
[0910] (5) ADCC Activity of Anti-CCR4 Chimeric Antibody Obtained
from GMD-Expressed Clone CHO/CCR4-LCA
[0911] ADCC activity of the purified anti-CCR4 chimeric antibody
obtained in the item 2(3) of Example 8 was measured in accordance
with the following method.
[0912] i) Preparation of Target Cell Suspension
[0913] CCR4/ELA cell described in WO01/64754 was prepared at
1.times.10 cells and 3.7 MBq equivalent of a radioactive substance
Na.sub.2.sup.51CrO.sub.4 was added to thereto, followed by reaction
at 37.degree. C. for 90 minutes to thereby label the cells with a
radioisotope. After the reaction, the cells were washed three times
by suspension in the RPMI1640-FBS(10) medium and subsequent
centrifugation, resuspended in the medium and then incubated at
4.degree. C. for 30 minutes on ice for spontaneous dissociation of
the radioactive substance. After centrifugation, the cells were
adjusted to 2.0.times.10.sup.5 cells/ml by adding 5 ml of the
RPMI1640-FBS(10) medium and used as a target cell suspension.
[0914] ii) Preparation of Effector Cell Suspension
[0915] From a healthy doner, 50 ml of venous blood was collected
and gently mixed with 0.5 ml of heparin sodium (manufactured by
Takeda Pharmaceutical). Using Lymphoprep (manufactured by Nycomed
Pharma AS), the mixture was centrifuged in accordance with the
manufacture's instructions to separate a mononuclear 4 cell layer.
The cells were washed three times by centrifuging with the
RPMI1640-FBS(10) medium and then resuspended in the medium to give
a density of 2.5.times.10.sup.6 cells/ml and used as a effector
cell suspension.
[0916] iii) Measurement of ADCC Activity
[0917] The target cell suspension prepared in the above i) was
dispensed at 50 .mu.l (1.times.10.sup.4 cells/well) into each well
of a 96 well U-bottom plate (manufactured by Falcon). Next, 100
.mu.l of the effector cell suspension prepared in the above ii) was
added thereto (2.times.10 cells/well, ratio of the effector cells
to the target cells was 25:1). Each of various anti-CCR4 chimeric
antibodies was further added thereto to give a final concentration
of 0.0025 to 2.5 .mu.l/ml, followed by reaction at 37.degree. C.
for 4 hours. After the reaction, the plate was centrifuged and the
amount .sup.51Cr in the supernatant was measured with a
.gamma.-counter. The amount of the spontaneously dissociated
.sup.51Cr was calculated by carrying out the same procedure using
the medium alone instead of the ejector cell suspension and
antibody solution, and measuring the amount .sup.5Cr in the
supernatant. The amount of the total dissociated .sup.51Cr was
calculated by carrying out the same procedure using the medium
alone instead of the antibody solution and adding 1 mol/L
hydrochloric acid instead of the effector cell suspension and
measuring the amount of .sup.51Cr in the supernatant. The ADCC
activity was calculated based on the above equation (1).
[0918] Results of the measurement of ADCC activity are shown in
FIG. 27. As shown in FIG. 27, ADCC activity of the purified
anti-CCR4 chimeric antibody obtained from the GMD-expressed clone
CHO/CCR4-LCA was decreased to a similar degree to that of the
KM3060 produced by the normal CHO cell-derived antibody-producing
clone obtain ed in Example 4. On the other hand, ADCC activity of
the purified anti-CCR4 chimeric antibody obtained from the pAGE249
vector-introduced clone CHO/CCR4-LCA showed a similar degree of
ADCC activity to that of the purified anti-CCR4 chimeric antibody
obtained from the clone CHO/CCR4-LCA. Based on the above results,
it was suggested that expression amount of GMD gene in the clone
CHO/CCR4-LCA is decreased and, as a result, an antibody having high
ADCC activity can be produced.
[0919] (6) Sugar Chain Analysis of Anti-CCR4 Chimeric Antibody
Derived from GMD-Expressed Clone CHO/CCR4-LCA
[0920] Sugar chains binding to the purified anti-CCR4 chimeric
antibody obtained in the item 2(3) of Example 8 were analyzed in
accordance with the method shown in the item 1 or Example 3, and
the analyzed results are shown in FIG. 28. In comparison with th
purified anti-CCR4 chimeric antibody prepared from the clone
CHO/CCR4-LCA in Example 7, the ratio of a sugar chain in which
1-position of fucose was not bound to 6-position of
N-acetylglucosamine in the reducing end through .alpha.-bond in the
purified anti-CCR4 chimeric antibody derived from the GMD
gene-expressed clone CHO/CCR4-LCA was decreased to 9% when
calculated from the peak area. Thus, it was shown that the ratio of
a sugar chain in which 1-position of fucose was not bound to
6-position of N-acetylglucosamine in the reducing end through
.alpha.-bond in the antibody produced by the cell is decreased to
similar level of the antibody produced by the clone 5-03.
EXAMPLE 9
[0921] Preparation of Anti-Fibroblast Growth Factor-8 Human
Chimeric Antibody
[0922] 1. Preparation of Cells Stably Producing Anti-Fibroblast
Growth Factor-8 Human Chimeric Antibody
[0923] Using a tandem type expression vector pKANTEX134 of an
anti-fibroblast growth factor-S (hereinafter referred to as
"FGF-S") human chimeric antibody described in Reference Example 3,
cells stably producing the anti-FGF-8 human chimeric antibody
(hereinafter referred to as "anti-FGF-8 chimeric antibody") was
prepared as follows.
[0924] (1) Preparation of Producing Cell Using Rat Myeloma YB2/0
Cell
[0925] After introducing 10 .mu.g of the anti-FGF-8 chimeric
antibody expression vector pKANTEX1334 into 4.times.10.sup.6 cells
of rat myeloma YB2/0 cell (ATCC CRL 1662) by electroporation
[Cytotechnology, 3, 133 (1990)], the cells were suspended in 40 ml
of Hybridoma-SFM-FBS(5) [Hybridoma-SFM medium (manufactured by
Invitrogen) containing 5% FBS (manufactured by PAA Laboratories)]
and dispensed at 200 .mu.l/well into a 96 well culture plate
(manufactured by Sumitomo Bakelite). After culturing at 37.degree.
C. for 24 hours in a 5% CO.sub.2 incubator, G418 was added to give
a concentration of 0.5 mg/ml, followed by culturing for 1 to 2
weeks. Culture supernatants were recovered from wells in which
colonies of transformants showing G418 resistance were formed and
their growth was confirmed, and antigen-binding activity of the
anti-FGF-8 chimeric antibody in the supernatants was measured by
the ELISA described in the item 2 of Example 9.
[0926] Regarding the transformants in wells in which production of
the anti-FGF-8 chimeric antibody was found in the culture
supernatants, in order to increase the antibody production amount
by using a dhfr gene amplification system, each of them was
suspended to give a density of 1 to 2.times.10.sup.5 cells/ml in
the Hybridoma-SFM-FBS(5) medium containing 0.5 mg/ml G418 and 50
nmol/l DHFR inhibitor MTX (manufactured by SIGMA) and dispensed at
1 ml into each well of a 24 well plate (manufactured by Greiner).
After culturing at 37.degree. C. for 1 to 2 weeks in a 5% CO.sub.2
incubator, transformants showing 50 nmol/l MTX resistance were
induced. Antigen-binding activity of the anti-FGF-8 chimeric
antibody in culture supernatants in wells where growth of
transformants was observed was measured by the ELISA described in
the item 2 of Example 9.
[0927] Regarding the transformants in wells in which production of
the anti-FGF-8 chimeric antibody was found in culture supernatants,
the MTX concentration was increased by a method similar to the
above to thereby finally obtain a transformant 5-D capable of
growing in the Hybridoma-SFM-FBS(5) medium containing 0.5 mg/ml
G418 and 200 nmol/l MTX and also highly producing the anti-FGF-8
chimeric antibody. The resulting transformant was subjected to
cloning by limiting dilution, and the resulting transformant cell
clone was named 5-D-10.
[0928] (2) Preparation of Producing Cell Using CHO/DG44 Cell
[0929] In accordance with the method described in Example 4, the
anti-FGF-8 chimeric antibody expression plasmid pKANTEX1334 was
introduced into CHO/DG44 cell and gene amplification was carried
out by using the drug MTX to obtain a transformant highly producing
the anti-FGF-8 chimeric antibody. The antibody expression amount
was measured using the ELISA described in the item 2 of Example 9.
The resulting transformant was cloned twice by limiting dilution,
and the resulting transformant cell clone was named 7-D-1-5.
[0930] 2. Binding Activity of Antibody to FGF-8 Partial Peptide
(ELISA)
[0931] Compound 2 (SEQ ID NO:21) was selected as a human FGF-8
peptide with which the anti-FGF-8 chimeric antibody can react. For
the activity measurement by the ELISA, a conjugate with BSA
(manufactured by Nacalai Tesque) was prepared by the following
method and used as the antigen. That is, 100 ml of a 25 mg/ml SMCC
[4-(N-maleimidomethyl)cyclohexane-1-ca- rboxylic acid
N-hydroxysuccinimide ester] (manufactured by SIGMA)-DMSO solution
was added dropwise to 900 ml of a PBS solution containing 10 mg of
BSA under stirring, followed by slowly stirred for 30 minutes. To a
gel filtration column such was NAP-10 column or the like which had
been equilibrated with 25 ml of PBS, 1 ml of the reaction solution
was applied, and the eluate eluted with 1.5 ml of PBS was used as a
BSA-SMCC solution (BSA concentration was calculated from A.sub.280
measurement). Next, 250 ml of PBS was added to 0.5 mg of Compound
2, 250 ml of DMF was added thereto and completely dissolved, and
then the above BSA-SMCC solution (1.25 mg as BSA) was added thereto
under stirring, followed by slow stirring for 3 hours. The reaction
solution was dialyzed against PBS at 4.degree. C. overnight, sodium
azide was added thereto to give a final concentration of 0.05% and
then filtered through a 0.22 .mu.m filter and used as a
BSA-compound 2 solution.
[0932] The conjugate prepared in the above was dispensed at 1
.mu.g/ml and 50 .mu.l/well into a 96 well plate for ELISA
(manufactured by Greiner) and adhered thereto by allowing it to
stand at 4.degree. C. overnight. After washing with PBS, 1% BSA-PBS
was added at 100 .mu.l/well and allowed to react at room
temperature for 1 hour to block the remaining active groups. After
washing each well with Tween-PBS, culture supernatant of a
transformant or a purified antibody was added at 50 .mu.l/well and
allowed to react at room temperature for 1 hour. After the reaction
and subsequent washing of each well with Tween-PBS, a
peroxidase-labeled goat anti-human IgG (.gamma.) antibody solution
(manufactured by American Qualex) diluted 3,000-fold with 1%
BSA-PBS was added as a secondary antibody solution at 50 .mu.l/well
and allowed to react at room temperature for 1 hour. After the
reaction and subsequent washing with Tween-PBS, the ABTS substrate
solution was added at 50 l/well to develop color, and the reaction
was stopped 10 minutes thereafter by adding 5% SDS solution at 50
.mu.l/well. Thereafter, OD415 was measured.
[0933] 3. Purification of Anti-FGF-8 Chimeric Antibody
[0934] (1) Culturing of YB2/0 Cell-Derived Producing Cell and
Purification of Antibody
[0935] The anti-FGF-8 chimeric antibody-expressing transformant 5-D
obtained in the item 1(1) of Example 9 was cultured in
Hybridoma-SFM (manufactured by Invitrogen) medium containing 200
nmol/l of MTX and 5% Daigo's GF21 (manufactured by Wako Pure
Chemical Industries) in a 182 cm.sup.2 flask (manufactured by
Greiner) at 37.degree. C. in a 5% CO.sub.2 incubator. After
culturing for 8 to 10 days, the anti-FGF-8 chimeric antibody was
purified from the culture supernatant recovered by using Prosep-A
(manufactured by Millipore) column and in accordance with the
attached manufacture's instructions. The purified anti-FGF-8
chimeric antibody was named YB2/0-FGF8 chimeric antibody.
[0936] (2) Culturing of CHO-DG44 Cell-Derived Antibody-Producing
Cells and Purification of Antibody
[0937] The anti-FGF-8 chimeric antibody-producing transformant cell
clone 7-D-I-5 obtained in the item 1(2) of Example 9 was cultured
in the IMDM-dFBS(10) medium in a 182 cm.sup.2 flask (manufactured
by Greiner) at 37.degree. C. in a 5% CO.sub.2 incubator. At the
stage where the cell density reached confluent several days
thereafter, the culture supernatant was discarded, the cells were
washed with 25 ml of PBS buffer and then 35 ml of EXCELL301 medium
(manufactured by JRH) was added thereto. After the culturing for 7
days at 37.degree. C. in a 5% CO.sub.2 incubator, the culture
supernatant was recovered. The anti-FGF-8 chimeric antibody was
purified from the culture supernatant by using Prosep-A
(manufactured by Millipore) column and in accordance with the
manufacture's instructions. The purified anti-FGF-8 chimeric
antibody was named CHO-FGF8 chimeric antibody.
[0938] When the binding activity of the YB2/0-FGF8 chimeric
antibody and CHO-FGF8 chimeric antibody to FGF-8 was measured by
the ELISA described in the item 2 of Example 9, they showed similar
binding activity.
[0939] 4. Analysis of Purified Anti-FGF-8 Chimeric Antibody
[0940] Each 4 .mu.g of the two anti-FGF-9 chimeric antibodies
produced by respective animal cells and purified in the item 3 of
Example 9 was subjected to SDS-PAGE according to a known method
[Nature, 227, 680 (1970)] and the molecular weight and purity were
analyzed. In each of the purified anti-FGF-8 chimeric antibodies, a
single band of about 150 Kd in molecular weight was found under
non-reducing conditions and two bands of about 50 Kd and about 25
Kd were found under reducing conditions. These molecular weights
almost coincided with the molecular weights deduced from the cDNA
nucleotide sequences of the antibody H chain and L chain (H chain:
about 49 Kd, L chain: about 23 Kd, whole molecule: about 144 Kd),
and also coincided with the reports showing that the IgG type
antibody shows a molecular weight of about 150 Kd under
non-reducing conditioned is degraded into H chain having a
molecular weight of about 50 Kd and L chain having a molecular
weight of about 25 Kd under reducing conditions due to cleavage of
the intramolecular S--S bond (Antibodies, Chapter 14 (1988);
Monoclonal Antibodies). Thus it was confirmed that the anti-FGF-8
chimeric antibody was purified as antibody molecules having correct
structures.
[0941] 5. Sugar Chain Analysis of Purified Anti-FGF-8 Chimeric
Antibodies
[0942] Sugar chain analysis of the YB2/0-FGF8 chimeric antibody
which is YB2/0 cell-derived anti-FGF-8 chimeric antibody and
anti-FGF-8 chimeric antibody CHO-FGF8 chimeric antibody which is
CHO/DG44 cell-de rived prepared in the item 4 of Example 9 was
carried out in accordance with the method described in the item 1
of Example 3. As a result, the ratio of a sugar chain in which
1-position of fucose was not bound to 6-position of
N-acetylglucosamine in the reducing end through .alpha.-bond were
58% in the YB2/0-FGF8 chimeric antibody and 13% in the CHO-FGF8
chimeric antibody. Hereinafter, these samples are called anti-FGF-8
chimeric antibody (58%) and anti-FGF-8 chimeric antibody (13%).
EXAMPLE 10
[0943] Preparation of Soluble Human Fc.gamma.RIIIa Protein
[0944] 1. Construction of a Soluble Human Fc.gamma.RIIIa Protein
Expression Vector
[0945] (1) Preparation of Human Peripheral Blood Monocyte cDNA
[0946] From a healthy doner, 30 ml of vein blood was collected,
gently mixed with 0.5 ml of heparin sodium (manufactured by Shimizu
Pharmaceutical) and then mixed with 30 ml of physiological saline
(manufactured by Otsuka Pharmaceutical). After the mixing, 10 ml of
each mixture was gently overlaid on 4 ml of Lymphoprep
(manufactured by NYCOMED PHARMA AS) and centrifuged at 2,000 rpm
for 30 minutes at room temperature. The separated monocyte
fractions in respective centrifugation tubes were combined and
suspended in 30 ml of RPMI1640-FBS(10). After centrifugation at
room temperature and at 1,200 rpm for 15 minutes, the supernatant
was discarded and the cell were suspended in 20 ml of
RPMI1640-FBS(10). This washing operation was repeated twice and
then 2.times.10.sup.6 cells/ml of peripheral blood monocyte
suspension was prepared using RPMI1640-FBS(10).
[0947] After 5 ml of the resulting peripheral blood monocyte
suspension was centrifuged at room temperature and at 800 rpm for 5
minutes in 5 ml of PBS, the Supernatant was discarded and the
residue was suspended in 5 mL of PBS. After centrifugation at room
temperature and at 800 rpm for 5 minutes, the supernatant was
discarded and total RNA was extracted by QIAamp RNA Blood Mini Kit
(manufactured by QIAGEN) and in accordance with the manufacture's
instructions.
[0948] A single-stranded cDNA was synthesized by reverse
transcription reaction to 2 .mu.g of the resulting total RNA, in a
series of 40 .mu.l containing oligo(dT) as primers using
SUPERSCRITP.TM. Preamplification System for First Strand cDNA
Synthesis (manufactured by Life Technologies) according to the
manufacture's instructions.
[0949] (2) Preparation Method of cDNA Encoding Human Fc.gamma.RIIIa
Protein
[0950] A cDNA encoding a human Fc.gamma.RIIIa protein (hereinafter
referred to as "hFc.gamma.RIIIa") was prepared as follows.
[0951] First, a specific forward primer containing a translation
initiation codon (represented by SEQ ID NO:22) and a specific
reverse primer containing a translation termination codon
(represented by SEQ ID NO:26) were designed from the nucleotide
sequence of hFc.gamma.RIIIa cDNA [J. Exp. Med. 170, 481
(1989)].
[0952] Next, using a DNA polymerase ExTaq (manufactured by Takara
Shuzo), 50 .mu.l of a reaction solution [1.times. concentration
ExTaq buffer (manufactured by Takara Shuzo), 0.2 mmol/l dNTPs, 1
.mu.mol/l of the above gene-specific primers (SEQ ID NOs:22 and
26)] containing 5 .mu.l of 20-fold diluted solution of the human
peripheral blood monocyte-derived cDNA solution prepared in the
item 1(1) of Example 10 was prepared, and PCR was carried out. The
PCR was carried out by 35 cycles of a reaction at 94.degree. C. for
30 seconds, at 56.degree. C. for 30 seconds and at 72.degree. C.
for 60 seconds as one cycle.
[0953] After the PCR, the reaction solution was purified by using
QIAquick PCR Purification Kit (manufactured by QIAGEN) and
dissolved in 20 .mu.l of sterile water. The products were digested
with restriction enzymes EcoRI (manufactured by Takara Shuzo) and
BamHI (manufactured by Takara Shuzo) and subjected to 0.8% agarose
gel electrophoresis to recover about 800 bp of a specific
amplification fragment.
[0954] On the other hand, 2.5 .mu.g of a plasmid pBluescript II
SK(-) (manufactured by Stratagene) was digested with restriction
enzymes EcoRI (manufactured by Takara Shuzo) and BamHI
(manufactured by Takara Shuzo), and digested products were
subjected to 0.8% agarose gel electrophoresis to recover a fragment
of about 2.9 kbp.
[0955] The human peripheral blood monocyte cDNA-derived
amplification fragment and plasmid pBluescript II SK(-)-derived
fragment obtained in the above were ligated by using DNA Ligation
Kit Ver. 2.0 (manufactured by Takara Shuzo). The strain Escherichia
coli DH5.alpha. (manufactured by TOYOBO) was transformed by using
the reaction solution, and a plasmid DNA was isolated from each of
the resulting ampicillin-resistant colonies according to a known
method.
[0956] A nucleotide sequence of the cDNA inserted into each plasmid
was determined by using DNA Sequencer 377 (manufactured by Parkin
Elmer) and BigDye Terminator Cycle Sequencing FS Ready Reaction Kit
(manufactured by Parkin Elmer) according to the manufacture's
instructions. It was confirmed that all of the inserted cDNAs whose
sequences were determined by this method encodes a complete ORF
sequence of the cDNA encoding hFc.gamma.RIIIa. A plasmid DNA
containing absolutely no reading error of bases in the sequence
accompanied by PCR was selected from them. Hereinafter, the plasmid
is called pBSFc.gamma.RIIIa5-3.
[0957] The thus determined full length cDNA sequence for
hFc.gamma.RIIIa is represented by SEQ ID NO:27, and its
corresponding amino acid sequence is shown in by SEQ ID NO:28.
[0958] (3) Preparation of a cDNA Encoding Soluble
hFc.gamma.RIIIa
[0959] A cDNA encoding soluble hFc.gamma.RIIIa (hereinafter
referred to as "shFc.gamma.RIIIa") having the extracellular region
of hFc.gamma.RIIIa (positions 1 to 193 in SEQ ID NO:28) and a
His-tag sequence at the C-terminal was constructed as follows.
[0960] First, a primer FcgR3-1 (represented by SEQ ID NO. 29)
specific for the extracellular region was designed from the
nucleotide sequence of hFc.gamma.RIIIa cDNA (represented by SEQ ID
NO:27).
[0961] Next, using a DNA polymerase ExTaq (manufactured by Takara
Shuzo), 50 .mu.l of a reaction solution [1.times. concentration
ExTaq buffer (manufactured by Takara Shuzo), 0.2 mmol/l dNTPs, 1
.mu.mol/l of the primer FcgR3-1, 1 .mu.mol/l of the primer M13M4
(manufactured by Takara Shuzo)) containing 5 ng of the plasmid
pBSFc.gamma.RIIIa5-3 prepared in the item 1(2) of Example 10 was
prepared, and PCR was carried out. The PCR was carried out by 35
cycles of a reaction at 94.degree. C. for 30 seconds, at 56.degree.
C. for 30 seconds and at 72.degree. C. for 60 seconds as one
cycle.
[0962] After the PCR, the reaction solution was purified by using
QIAquick PCR Purification Kit (manufactured by QIAGEN) and
dissolved in 20 .mu.l of sterile water. The products were digested
with restriction enzymes PstI (manufactured by Takara Shuzo) and
BamHI (manufactured by Takara Shuzo) and subjected to 0.8% agarose
gel electrophoresis to recover about 110 bp of a specific
amplification fragment.
[0963] On the other hand, 2.5 g of the plasmid pBSFc.gamma.RIIIa5-3
was digested with restriction enzymes PstI (manufactured by Takara
Shuzo) and BamHI (manufactured by Takara Shuzo), and the digested
products were subjected to 0.8% agarose gel electrophoresis to
recover a fragment of about 3.5 kbp.
[0964] The hFc.gamma.RIIIa cDNA-derived amplification fragment and
plasmid pBSFc.gamma.RIIIa5-3-derived fragment obtained in the above
were ligated by using DNA Ligation Kit Ver. 2.0 (manufactured by
Takara Shuzo). The strain Escherichia coli DH5.alpha. (manufactured
by TOYOBO) was transformed by using the reaction solution, and a
plasmid DNA was isolated from each of the resulting
ampicillin-resistant colonies according to a known method.
[0965] A nucleotide sequence of the cDNA inserted into each plasmid
was determined by using DNA Sequencer 377 (manufactured by Parkin
Elmer) and BigDye Terminator Cycle Sequencing FS Ready Reaction Kit
(manufactured by Parkin Elmer) according to the manufacture's
instructions. It was confirmed that all of the inserted cDNAs whose
sequences were determined by this method encodes a complete ORF
sequence of the cDNA encoding shFc.gamma.RIIIa of interest. A
plasmid DNA containing absolutely no reading error of bases in the
sequence accompanied by PCR was selected from them. Hereinafter,
this plasmid is named pBSFc.gamma.RIIIa+His3.
[0966] The thus determined full length cDNA sequence for
shFc.gamma.RIIIa is represented by SEQ ID NO:30, and its
corresponding amino acid sequence is represented by SEQ ID
NO:31.
[0967] (4) Construction of shFc.gamma.RIIIa Expression Vector
[0968] The shFc.gamma.RIIIa expression vector was constructed as
follows.
[0969] After 3.0 .mu.g of the plasmid pBSFc.gamma.RIIIa+His3
obtained in the item 1(3) of Example 10 was digested with
restriction enzymes EcoRI (manufactured by Takara Shuzo) and BamHI
(manufactured by Takara Shuzo), the digested products were
subjected to 0.8% agarose gel electrophoresis to recover a fragment
of about 620 bp.
[0970] Separately, 2.0 .mu.g of the plasmid pKANTEX93 described in
WO97/10354 was digested with restriction enzymes EcoRI
(manufactured by Takara Shuzo) and BamHI (manufactured by Takara
Shuzo), and the digested products were subjected to 0.8% agarose
gel electrophoresis to recover a fragment of about 10.7 kbp.
[0971] The DNA fragment containing shFc.gamma.RIIIa cDNA and the
plasmid pKANTEX93-derived fragment obtained in the above were
ligated by using DNA Ligation Kit Ver. 2.0 (manufactured by Takara
Shuzo). The strain Escherichia coli DH5.alpha. (manufactured by
TOYOBO) was transformed by using the reaction solution, and a
plasmid DNA was isolated from each of the resulting
ampicillin-resistant colonies according to a known method.
[0972] A nucleotide sequence of the cDNA inserted into each plasmid
was determined by using DNA Sequencer 377 (manufactured by Parkin
Elmer) and BigDye Terminator Cycle Sequencing FS Ready Reaction Kit
(manufactured by Parkin Elmer)+, in accordance with the manual
attached thereto. It was confirmed that all of the plasmids whose
sequences were determined by this method encodes the cDNA of
interest encoding shFc.gamma.RIIIa. Hereinafter, the obtained
expression vector was named pKANTEXFc.gamma.RIIIa-His.
[0973] 2. Preparation of Cell Stably Producing shFc.gamma.RIIIa
[0974] Cells stably producing shFc.gamma.RIIIa were prepared by
introducing the shFc.gamma.RIIIa expression vector
pKANTEXFc.gamma.RIIIa-His constructed in the item 1 of Example 10
into rat myeloma YB2/0 cell (ATCC CRL-1662, J. Cell. Biol., 93, 576
(1982)],
[0975] pKANTEXFc.gamma.RIIIa-His was digested with a restriction
enzyme AatII to obtain a linear fragment, 10 .mu.g thereof was
introduced into 4.times.10.sup.6 cells by electroporation
[Cytotechnology, 1, 133 (1990)], and the resulting cells were
suspended in 40 ml of Hybridoma-SFM-FBS(10) [Hybridoma-SFM medium
(manufactured by Life Technologie) containing 10% FBS] and
dispensed at 200 .mu.l/well into a 96 well culture plate
(manufactured by Sumitomo Bakelite). After culturing at 37.degree.
C. for 24 hours in a 5% CO.sub.2 incubator, G418 was added to give
a concentration of 1.0 mg/ml, followed by culturing for 1 to 2
weeks. Culture supernatants were recovered from wells in which
colonies of transformants showing G418 resistance were formed and
their growth was confirmed, and expression amount of
shFc.gamma.RIIIa in the supernatants was measured by the ELISA
described in the item 3 of Example 10.
[0976] Regarding the transformants in wells in which expression of
the shFc.gamma.RIIIa was confirmed in the culture supernatants, in
order to increase the antibody production by using a dhfr gene
amplification system, each of them was suspended to give a density
of 1 to 2.times.10 cells/ml in the Hybridoma-SFM-FBS(10) medium
containing 1.0 mg/ml G418 and 50 nmol/l DHFR inhibitor MTX
(manufactured by SIGMA) and dispensed at 2 ml into each well of a
24 well plate (manufactured by Greiner). After culturing at
37.degree. C. for 1 to 2 weeks in a 5% CO.sub.2 incubator,
transformants showing 50 nmol/l MTX resistance were induced.
Expression level of shFc.gamma.RIIIa in culture supernatants in
wells where growth of transformants was observed was measured by
the ELISA described in The item 3 of Example 10. Regarding the
transformants in wells in which expression of the shFc.gamma.RIIIa
was found in culture supernatants, the MTX concentration was
increased to 100 nmol/l and then to 200 nmol/l sequentially by a
method similar to the above to thereby finally obtain a
transformant capable of growing in the Hybridoma-SFM-FBS(10) medium
containing 1.0 mg/ml G418 and 200 nmol/l MTX and also of highly
producing shFc.gamma.RIIIa. The resulting transformant was cloned
twice by limiting dilution. The obtained transformant was named
clone KC1107.
[0977] 3. Detection of shFc.gamma.RIIIa (ELISA)
[0978] shFc.gamma.RIIIa in culture supernatant or purified
shFc.gamma.RIIIa was detected or determined by the ELISA shown
below.
[0979] A solution of a mouse antibody against His-tag, Tetra-His
Antibody (manufactured by QIAGEN), adjusted to 5 .mu.g/ml with PBS
was dispensed at 50 .mu.l/well into each well of a 96 well plate
for ELISA (manufactured by Greiner) and allowed to react at
4.degree. C. for 12 hours or more. After the reaction, 1% BSA-PBS
was added at 100 .mu.l/well and allowed to react at room
temperature for 1 hour to block the remaining active groups. After
1% BSA-PBS was discarded, culture supernatant of the transformant
or each of various dilution solutions of purified shFc.gamma.RI Ira
was added at 50 .mu.l/well and allowed to react at room temperature
for 1 hour. After the reaction and subsequent washing of each well
with Tween-PBS, a biotin-labeled mouse anti-human CD16 antibody
solution (manufactured by PharMingen) diluted 50-fold with 1%
BSA-PBS was added at 50 .mu.l/well and allowed to react at room
temperature for 1 hour. After the reaction and subsequent washing
with Tween-PBS, a peroxidase-labeled Avidin D solution
(manufactured by Vector) diluted 4,000-fold with 1% BSA-PBS was
added at 50 .mu.l/well and allowed to react at room temperature for
1 hour. After the reaction and subsequent washing with Tween-PBS,
the ABTS substrate solution was added at 50 .mu.l/well to develop
color, and OD415 was measured.
[0980] 4. Purification of shFc.gamma.RIIa
[0981] The shFc.gamma.RIIIa-producing transformant cell clone
KC1107 obtained in the item 2 of Example 10 was suspended in
Hybridoma-SFM-GF(5) [Hybridoma-SFM medium (manufactured by Life
Technologic) containing 5% Daigo's GF21 (manufactured by Wako Pure
Chemical Industries)] to give a density of 3.times.10 cells/ml and
dispensed at 50 ml into 182 cm.sup.2 flasks (manufactured by
Greiner). After culturing at 37.degree. C. for 4 days in a 5%
CO.sub.2 incubator, the culture supernatants were recovered.
shFc.gamma.RIIIa was purified from the culture supernatants by
using Ni-NTA agarose (manufactured by QIAGEN) column according to
the manufacture's instructions.
[0982] 5. Analysis of Purified shFc.gamma.RIIIa
[0983] A concentration of purified shFc.gamma.RIIIa obtained in the
item 4 of Example 10 was calculated by amino acid composition
analysis as follows. A part of purified shFc.gamma.RIIIa was
suspended in 6 mol/l hydrochloric acid-1% phenol solution, and
hydrolysed in a gas phase at 110.degree. C. for 20 hours. Work
Station manufactured by Waters was used for the hydrolysis. Amino
acids after the hydrolysis were converted into PTC-amino acid
derivatives in accordance with the method of Bidlingmeyer et al.
[J. Chromatogr., 336, 93 (1984)] and analyzed by using PicoTag
Amino Acid Analyzer (manufactured by Waters).
[0984] Next, about 0.5 .mu.g of purified shFc.gamma.RIIIa was
subjected to SDS-PAGE under reducing conditions according to a
known method [Nature 227, 680 (1970)] to analyze its molecular
weight and purity. The results are shown in FIG. 6. As shown in
FIG. 29, a broad band of 36 to 38 Kd in molecular weight was
detected in purified can be bound are present in the extracellular
region of hFc.gamma.RIIIa [J. Exp. Med., 170, 481 (1989)], it was
considered that the broad molecular weight distribution of purified
shFc.gamma.RIIa is based on the irregularity of sugar chain
addition. On the other hand, when the N-terminal amino acid
sequence of purified shFc.gamma.RIIIa was analyzed by automatic
Edman degradation using a protein sequencer PPSQ-10 (manufactured
by Shimadzu), a sequence expected from the cDNA of shFc.gamma.RIIIa
was obtained, so that it was confirmed that shFc.gamma.RIIa of
interest was purified.
EXAMPLE 11
[0985] Evaluation of Binding Activity of Various Chimeric
Antibodies to shFc.gamma.RIIIa
[0986] 1. Evaluation of shFc.gamma.RIIIa-Binding Activity of
Anti-GD3 Chimeric Antibodies Having a Different Ratio of a Sugar
Chain in which 1-Position of Fucose was not Bound to 6-Position of
N-acetylglucosamine in the Reducing End through .alpha.-Bond
[0987] The shFc.gamma.RIIIa-binding activity of the anti-GD3
chimeric antibody (45%) and anti-GD3 chimeric antibody (7%)
described in the item 1 of Example 3 which are two anti-GD3
chimeric antibodies having a different ratio of a sugar chain in
which 1-position of fucose was not bound to 6-position of
N-acetylglucosamine in the reducing end through .alpha.-bond was
measured by ELISA as follows.
[0988] According to the method described in the item 3 of Example
1, GD3 was immobilized at 100 pmol/well on a 96 well plate for
ELISA (manufactured by Greiner). The 1% BSA-PBS was added at 100
.mu.l/well and allowed to react at room temperature for 1 hour to
block the remaining active groups. After washing each well with
Tween-PBS, a solution of each anti-GD3 chimeric antibody diluted
with 1% BSA-PBS was added at 50 .mu.l/well and allowed to react at
room temperature for 1 hour. After the reaction and subsequent
washing of each well with Tween-PBS, an shFc.gamma.RIIIa solution
prepared by diluting it to 2.3 .mu.g/ml with 1% BSA-PBS was added
at 50 .mu.l/well and allowed to react at room temperature for 1
hour. After the reaction and subsequent washing with Tween-PBS, a
solution of a mouse antibody against His-tag, Tetra-His Antibody
(manufactured by QIAGEN), adjusted to 1 .mu.g/ml with 1% BSA-PBS
was added at 50 .mu.l/well and allowed to react at room temperature
for 1 hour. After the reaction and subsequent washing with
Tween-PBS, a peroxidase-labeled goat anti-mouse IgG1 antibody
solution (manufactured by ZYMED) diluted 200-fold with 1% BSA PBS
was added at 50 .mu.l/well and allowed to react at room temperature
for 1 hour. After the reaction and subsequent washing with
Tween-PBS, the ABTS substrate solution was added at 50 .mu.l/well
to develop color, and OD415 was measured. In addition, it was
confirmed that there is no difference in the amount of the anti-GD3
chimeric antibodies bound to the plate by adding each of the
anti-GD3 chimeric antibodies to another plate and carrying out the
ELISA described in item 3 of Example 1. The results of the
measurement of the binding activity of the various anti-GD3
chimeric antibodies for shFc.gamma.RIIIa are shown in FIG. 30. As
shown in FIG. 30, regarding the binding activity of the anti-GD3
chimeric antibodies to shFc.gamma.RIIIa, the anti-GD3 chimeric
antibody (45%) having a high ratio of a sugar chain in which
1-position of fucose was not bound to 6-position of
N-acetylglucosamine in the reducing end through .alpha.-bond had 10
times or more higher activity.
[0989] 2. Evaluation of shFc.gamma.RIIIa-Binding Activity of
Anti-FGF-8 Chimeric Antibodies Having a Different Ratio of a Sugar
Chain in which 1-Position of Fucose was not Bound to 6-Position of
N-acetylglucosamine in the Reducing End through .alpha.-Bond
[0990] The shFc.gamma.RIIa-binding activity of the anti-FGF-8
chimeric antibody (58%) and anti-FGF-8 chimeric antibody (13%)
described in the item 5 of Example 9 which were as two anti-FGF-8
chimeric antibodies having different ratio of a sugar chain in
which 1-position of fucose was not bound to 6-position of
N-acetylglucosamine in the reducing end through .alpha.-bond was
measured by ELISA as follows.
[0991] The human FGF-8 peptide conjugate prepared in the item 2 of
Example 9 at a concentration of 1.0 .mu.g/ml was dispensed at 50
.mu.l/well into a 96-well plate for ELISA (manufactured by Greiner)
and adhered thereto by allowing it to stand at 4.degree. C.
overnight. After washing with PBS, 1% BSA-PBS was added at 100
.mu.l/well and allowed to react at room temperature for 1 hour to
block the remaining active groups. After washing each well with
Tween-PBS, a solution of each anti-FGF-8 chimeric antibody diluted
with 1% BSA-PBS was added at 50 .mu.l/well and allowed to react at
room temperature for 1 hour. After the reaction and subsequent
washing of each well with Tween-PBS, an shFc.gamma.RIIIa solution
prepared by diluting it to 3.0 .mu.g/ml with 1% BSA-PBS was added
at 50 .mu.l/well and allowed to react at room temperature for 1
hour. After the reaction and subsequent washing with Tween-PBS, a
solution of a mouse antibody against His-tag, Tetra-His Antibody
(manufactured by QIAGEN), adjusted to 1 .mu.g/ml with 1% BSA-PBS,
was added at 50 .mu.l/well and allowed to react at room temperature
for 1 hour. After the reaction and subsequent washing with
Tween-PBS, a peroxidase-labeled goat anti-mouse IgG1 antibody
solution (manufactured by ZYMED) diluted 200-fold with 1% BSA-PBS
was added at 50 .mu.l/well and allowed to react at room temperature
for 1 hour. After the reaction and subsequent washing with
Tween-PBS, the ABTS substrate solution was added at 50 .mu.l/well
to develop color, and OD415 was measured. In addition, by adding
each of the anti-FGF-8 chimeric antibodies to another plate and
carrying out the ELISA described in the item 2 of Example 9, it was
confirmed that there is no difference in the amount of the various
anti-FGF-8 chimeric antibodies bound to the plate. The results of
the measurement of the binding activity of the various anti-FGF-8
chimeric antibodies for shFc.gamma.RIIIa are shown in FIG. 31. As
shown in FIG. 31, regarding the binding activity of the anti-FGF8
chimeric antibodies to shFc.gamma.RIIIa the anti-FGF-8 chimeric
antibody (58%) having a high ratio of a sugar chain in which
1-position of fucose was not bound to 6-position of
N-acetylglucosamine in the reducing end through .alpha.-bond had
100 times or more higher activity.
[0992] 3. Evaluation of shFc.gamma.RIIIa-Binding Activity of
Anti-CCR4 Chimeric Antibodies Having a Different Ratio of a Sugar
Chain in which 1-Position of Fucose is not Bound to 6-Position of
N-acetylglucosamine in the Reducing End through .alpha.-Bond
[0993] The shFc.gamma.RIIIa-binding activity of the seven anti-CCR4
chimeric antibodies described in the item 5 of Example 4, having a
different ratio of a sugar chain in which 1-position of fucose was
not bound to 6-position of N-acetylglucosamine in the reducing end
through .alpha.-bond, was measured by ELISA as follows.
[0994] The human CCR4 extracellular peptide conjugate prepared in
the item 2 of Example 4 at a concentration of 1.0 .mu.g/ml was
dispensed at 50 .mu.l/well into a 96 well plate for ELISA
(manufactured by Greiner) and adhered thereto by allowing it to
stand at 4.degree. C. overnight. After washing with PBS, 1% BSA-PBS
was added at 100 .mu.l/well and allowed to react at room
temperature for 1 hour to block the remaining active groups. After
washing each well with Tween-PBS, a solution of each anti-CCR4
chimeric antibody diluted with 1% BSA-PBS was added at 50
.mu.l/well and allowed to react at room temperature for 1 hour.
After the reaction and subsequent washing of each well with
Tween-PBS, an shFc.gamma.RIIIa solution prepared by diluting it to
3.0 .mu.g/ml with 1% BSA-PES was added at 50 .mu.l/well and allowed
to react at room temperature for 1 hour. After the reaction and
subsequent washing with Tween-PBS, a solution of a mouse antibody
against His-tag, Tetra-His Antibody (manufactured by QIAGEN),
adjusted to 1 .mu.g/ml with 1% BSA-PBS, was added at 50 .mu.l/well
and allowed to react at room temperature for 1 hour. After the
reaction and subsequent washing with Tween-PBS, a
peroxidase-labeled goat anti-mouse IgG1 antibody solution
(manufactured by ZYMED) diluted 200-fold with 1% BSA-PBS was added
at 50 .mu.l/well and allowed to react at room temperature for 1
hour. After the reaction and subsequent washing with Tween-PBS, the
ABTS substrate solution was added at 50 .mu.l/well to develop
color, and OD415 was measured. In addition, it was confirmed that
there is no difference in the amount of the anti-CCR4 chimeric
antibodies bound to the plate by adding each of the anti-CCR4
chimeric antibodies to another plate and carrying out the ELISA
described in the item 2 of Example 4. The results of the
measurement of the binding activity of the various anti-CCR4
chimeric antibodies to shFc.gamma.RIIIa are shown in FIG. 32A. As
shown in FIG. 32A, the binding activity of the anti-CCR4 chimeric
antibodies to shFc.gamma.RIIIa increased in proportion to the ratio
of a sugar chain in which 1-position of fucose was not bound to
6-position of N-acetylglucosamine in the reducing end through
.alpha.-bond. FIG. 32B shows a plotted graph on a relationship
between the ratio of a sugar chain in which 1-position of fucose
was not bound to 6-position of N-acetylglucosamine in the reducing
end through .alpha.-bond, at antibody concentrations of 4 .mu.g/ml
and 40 .mu.g/ml (the abscissa) and the shFc.gamma.RIIIa-binding
activity (the ordinate). As shown in FIG. 32B, the
shFc.gamma.RIIIa-binding activity was hardly detected in the
anti-CCR4chimeric antibody (8%), anti-CCR4 chimeric antibody (9%)
and anti-CCR4 chimeric antibody (18%), as antibodies having 20% or
less of the ratio of a sugar chain in which 1-position of fucose
was not bound to 6-position of N-acetylglucosamine in the reducing
end through .alpha.-bond.
[0995] The above results clearly show that antibodies having a
sugar chain in which 1-position of fucose is not bound to
6-position of N-acetylglucosamine in the reducing end through
.alpha.-bond have higher shFc.gamma.RIIIa-bonding activity than
antibodies having a .alpha.1,6-fucose sugar chain. In addition,
since antibodies having a sugar chain in which 1-position of fucose
is not bound to 6-position of N-acetylglucosamine in the educing
end through .alpha.-bond has higher ADCC activity than antibodies
having a .alpha.1,6-fucose sugar chain as shown in Examples 3 and
4, it was strongly suggested that the high ADCC act ivity of
antibodies a sugar chain in which 1-position of fucose is not bound
to 6-position of N-acetyl glucosamine in the reducing end through
.alpha.-bond is based on the high shFc.gamma.RIIIa-binding
activity. As shown in FIG. 32B, the proportion between
Fc.gamma.RIIIa binding activity and the ratio of a sugar chain in
which 1-position of fucose is not bound to 6-position of
N-acetylglucosamine in the reducing end through .alpha.-bond is
found. Therefore, Fc.gamma.RIIIa-binding activity can be measured
and the ratio of a sugar chain in which 1-position of fucose is not
bound to 6-position of N-acetylglucosamine in the reducing end
through .alpha.-bond can be determined by constructing such a
calbiation curve in advance. By using the above method, the
cyototoxic activity can be prospected easily without determining
the cytotoxic activity of the antibody composition.
EXAMPLE 12
[0996] Evaluation of Antibody Produced by Lectin-Resistant CHO/DG44
Cells to shFc.gamma.RIIIa
[0997] Binding activities of the anti-CCR4 chimeric antibody
produced by the lectin-resistant clone CHO/CCR4-LCA purified in the
item 3 of Example 7 [hereinafter referred to as anti-CCR4 chimeric
antibody (48%)"), the anti-CCR4 chimeric antibody KM2760-1 produced
by a YB2/0 cell-derived antibody-producing clone purified in the
item 3 of Example 4 [anti-CCR4 chimeric antibody (87%)] and the
anti-CCR4 chimeric antibody KM3060 produced by a CHO/DG44
cell-derived antibody-producing clone 5-03 (anti-CCR4 chimeric
antibody (8%)] to shFc.gamma.RIIIa were measured according to the
method described in the item 3 of Example 11. As a result, as shown
in FIG. 33, the anti-CCR4 chimeric antibody (48%) produced by the
lectin-resistant clone CHO/CCR4-LCA showed 100 times or more higher
binding activity to shFc.gamma.RIIIa than the anti-CCR4 chimeric
antibody (8%) produced by the clone 5-03. Also, this activity was
about 1/3 of the anti-CCR4 chimeric antibody (87%) produced by the
YB2/0 cell-derived antibody-producing clone.
[0998] The above results clearly show that an antibody having 100
times or more higher binding activity to shFc.gamma.RIIIa can be
prepared by using lectin-resistant CHO/DG44 cell than by using
CHO/DG44 cell which is its parent cell.
EXAMPLE 13
[0999] Method for Screening Antibody Composition Having High ADCC
Activity Based on Binding Activity to shFc.gamma.RIIIa
[1000] A anti-GD3 chimeric antibody expression plasmid pCHi641LHGM4
was introduced into the LCA-resistant clone CHO-LCA obtained in the
item 1 of Example 7 according to the method described in the item
2(2) of Example 1, and gene amplification was carried out by using
MTX to produce a transformant producing an anti-GD3 chimeric
antibody. Cloning was carried out by limiting dilution method using
the obtained transformant to obtain several clones. After each
clone was cultured, the culture medium was recovered when it became
confluent. A concentration of the anti-GD3 chimeric antibody in the
culture supernatant way diluted to 1 .mu.g/ml, and the
shFc.gamma.RIIIa-binding activity was measured using the diluted
antibody solution by the ELISA described in the item 1 of Example
11. At the same time, solutions of the anti-GD3 chimeric antibody
produced by a YB2/0 cell-derived antibody-producing clone and the
anti-GD3 chimeric antibody produced by a CHO/DG44 cell-derived
antibody-producing clone, purified in the item 4 of Example 1 were
prepared by diluting them to 1 .mu./ml, and their
shFc.gamma.RIIIa-binding activities were also measured.
[1001] Based on the measured results, a transformant cell clone
capable of producing an antibody showing the activity equal to or
higher than the binding activity of the anti-GD3 chimeric antibody
produced by the CHO/DG44 cell-derived antibody-producing clone and
also equal to or lower than the binding activity of the anti-GD3
chimeric antibody produced by the YB2/0 cell-derived
antibody-producing clone was selected.
[1002] A purified antibody was obtained from the culture
supernatant by culturing the selected transformant cell clone
according to the method described in the item 4(2) of Example 1.
When monosaccharide composition analysis of the purified antibody
was carried out according to the method described in the item 1 of
Example 3, the ratio of a sugar chain in which 1-position of fucose
was not bound to 6-position of N-acetylglucosamine in the reducing
end through .alpha.-bond was 42%. Hereinafter, this sample is
called anti-GD3 chimeric antibody (42%). When antigen-binding
activity of the purified anti-GD3 chimeric antibody (42%) was
evaluated by the ELISA described in the item 3 of Example m, it was
equivalent to those of the anti-GD3 chimeric antibody produced by a
YB2/0 cell-derived antibody-producing clone and the anti-GD3
chimeric antibody produced by a CHO/DG44 cell-derived
antibody-producing clone purified in the item 4 of Example 1. In
addition, the ADCC activity of each anti-GD3 chimeric antibody was
evaluated according to the method described in the item 2 of
Example 2. For comparison, the ADCC activity of a sample of the
anti-GD3 chimeric antibody produced by a CHO/DG44 cell-derived
antibody-producing clone, wherein the ratio of a sugar chain in
which 1-position of fucose was not bound to 6-position of
acetylglucosamine in the reducing end through .alpha.-bond was 12%
as a result of its monosaccharide composition analysis [hereinafter
named "anti-GD3 chimeric antibody (12%)"] was measured. The results
are shown in FIG. 34. In comparison with the anti-GD3 chimeric
antibody (12%) produced by a CHO/DG44 cell-derived
antibody-producing clone, about 30 times increase in the ADCC
activity was observed in the anti-GD3 chimeric antibody (42%)
produced by an antibody-producing clone selected based on the high
binding activity to shFc.gamma.RIIIa.
[1003] Based on the above results, it was found that an antibody
composition having high ADCC activity can be screened by screening
an antibody composition having high binding activity to
shFc.gamma.RIIIa.
EXAMPLE 14
[1004] Preparation of FGF-8b/Fc Fusion Protein.
[1005] 1. Construction of the Expression Vector of FGF-8b/Fc Fusion
Protein
[1006] A humanized antibody expression vector pKANTEX93 [Mol.
Immunol., 37, 1035 (2000)] was digested with restriction enzymes
ApaI and BamHI and a fragment containing about 1.0 kbp of human
IgG1 subclass CH (hC.gamma.1) was obtained by using QIAquick Gel
Extraction Kit (manufactured by QIAGEN). A plasmid pBluescript II
SK(-) (manufactured by STRATAGENE) was also digested with similar
restriction enzymes to obtain a fragment of about 2.9 kbp. The
fragments were ligated using Solution I of TAKARA DNA Ligation Kit
Ver. 2 (manufactured by Takara Shuzo) and E. coli DH5.alpha.
(manufactured by TOYOBO) was transformed to construct a plasmid
phC.gamma.1/SK (-).
[1007] In order to ligate hC.gamma. 1 with cDNA of FGF8, a
synthetic DNA having the nucleotide sequence represented by SEQ ID
NO:86 was designed. The synthetic DNA contains plural restriction
enzyme recognition sequences at its 5' terminal for cloning to
pBluescript II SK(-), and the synthesis of the DNA was consigned to
Proligo. To 50 .mu.l of a solution containing EX Taq Buffer
(Mg.sup.2+ plus) in TaKaRa Ex Taq (manufactured by Takara Shuzo) at
1.times.concentration, 1 ng of plasmid phC.gamma. 1/SK (-), 0.25
mmol/L dNTPs, 0.5 .mu.mol/l of the synthetic DNA having the
nucleotide sequence represented by SEQ ID N0.86, 0.5 .mu.mol/L M13
primer RV and 1.25 unit of TaKaRa Ex Taq were added, and the
resulting solution was subjected to 30 cycles of heating at
94.degree. C. for 30 minutes, 56.degree. C. for 30 minutes and
72.degree. C. for 1 minute as one cycle by using DNA thermal cycler
GeneAmp PCR System 9700 (manufactured by PERKIN ELMER). A PCR
amplified fragment was purified from the total reaction solution by
using QIAquick PCR purification Kit (manufactured by QIAGEN). Then,
the purified fragment was digested with restriction enzymes KpnI
and BamHI to obtain a fragment of about 0.75 kbp. In addition, the
pBluescript II SK (-) (manufactured by STRATAGENE) was digested
with similar restriction enzymes to obtain a fragment of about 2.9
kbp. These fragments were ligated by using Ligation high
(manufactured by TOYOBO) and E. coli DH5.alpha. (manufactured by
TOYOBO) was transformed to construct a plasmid
p.DELTA.hC.gamma.1/SK(-).
[1008] Using the FGF-8b gene cloned plasmid pSC17 [Proc. Natl.
Acad. Sci., 89, 8928 (1992)] as the template, PCR was carried out
as follows to obtain a structure gene region fragment of FGF-8b. To
50 .mu.l of a solution containing EX Taq Buffer (Mg.sup.2, plus) in
TaKaRa Ex Taq (manufactured by Takara Shuzo) at 1.times.
concentration, 1 ng of plasmid pSC17, 0.25 mmol/l dNTPs, 10
.mu.mol/l of synthetic DNAs having the nucleotide sequence
represented by SEQ ID NOs:87 and 88 and 2.5 unit of TaKaRa Ex Taq
were added, and the resulting solution was subjected to 35 cycles
of heating at 94.degree. C. for 1 minute, 55.degree. C. for 1
minute and 72.degree. C. for 2 minutes as one cycle, followed by
heating at 72.degree. C. for 10 minutes, by using DNA thermal
cycler GeneAmp PCR System 9700 (manufactured by PERKIN ELMER). A
PCR amplified fragment was purified from the total reaction
solution, and the purified fragment was digested with restriction
enzymes EcoRI and BamHI to obtain a fragment of about 0.66 kbp.
Also, plasmid pBluescript II SK(-) (manufactured by STRATAGENE) was
digested with similar restriction enzymes to obtain a fragment of
about 2.9 kbp. Then, the fragments were ligated by using T4 DNA
Ligase (manufactured by Takara Shuzo) and E. coli DH5.alpha.
(manufactured by TOYOBO) was transformed to construct a plasmid
pFGF-8b/SK(-).
[1009] Next, the plasmid p.DELTA.hC.gamma. 1/SK(-) was digested
with restriction enzymes ApaI and EcoRI to obtain a fragment of
about 3.7 kbp. In addition, the plasmid pFGF-8b/SK(-) was digested
with similar restriction enzymes to obtain a fragment of about 0.6
kbp. Then, the fragments were ligated by using Ligation high
(manufactured by TOYOBO) and E. coli DH5.alpha. (manufactured by
TOYOBO) was transformed to construct a plasmid
pFGF8b+hIgG/SK(-).
[1010] Next, the thus constructed plasmid pFGF8b+hIgG/SK (-) was
digested with restriction enzymes EcoRI and BamHI, a fragment of
about 1.34 kbp was obtained. Also, pKANTEX93 was digested with
similar restriction enzymes to obtain a fragment of about 8.8 kbp.
Then, the fragments were ligated by using Ligation high
(manufactured by TOYOBO) and E. coli DH5 D (manufactured by TOYOBO)
was transformed to construct an expression vector pKANTEX/FGF8Fc
for animal cell containing cDNA of FGF8b-Fc fusion protein
represented by SEQ ID NO:89.
[1011] 2. Stable Expression Using Animal Cell of FGF-8b/Fc Fusion
Protein
[1012] A stable expression clone of FGF-8/Fc fusion protein was
prepared by introducing the FGF-8 fusion protein expression vector
pKANTEX/FGF8Fc for animal cell which was constructed in the item 1
of Example 14 into various cells, and selecting a suitable
clone.
[1013] (1) Preparation of Producing Cell Using Rat Myeloma YB2/0
Cells
[1014] After 10 .mu.g of the FGF8-Fc fusion protein expression
vector pKANTEX/FGF8Fc was introduced into 4.times.10.sup.6 cells of
rat myeloma YB2/0 cell by electroporation, the resulting cells were
suspended in 20 to 40 ml of RPMI1640-FBS(10) and the solution was
dispensed at 200 .mu.l/well into a 96-well culture plate
(manufactured by Sumitomo Bakelite). After culturing at 37.degree.
C. for 24 hours in a 5% CO.sub.2 incubator, G418 was added thereto
to give a concentration of 0.5 ml/ml, followed by culturing for 1
to 2 weeks. The culture supernatant was recovered from wells in
which colonies of G418 resistance transformants showing were formed
and growth of colonies was observed, and the binding activity of
the FGF8-Fc fusion protein in the supernatant to an anti-FGF-8
antibody was measured by the ELISA described in the item 4 of
Example 14. As the anti-FGF-8 antibody, KM1334 (U.S. Pat. No.
5,952,472) was used.
[1015] Regarding the transformants in wells in which production of
the FGF-8/Fc protein was observed in culture supernatants, in order
to increase the production amount of the fusion protein by using a
dhfr gene amplification system, each of them was suspended in the
Hybridoma-SFM-FBS(S) medium comprising 0.5 mg/ml G418 and 50 nmol/L
DHFR inhibitor, MTX (manufactured by SIGMA), and dispensed into
each well of a 24 well plate for expansion culturing. After
culturing at 37.degree. C. for 1 to 2 weeks in a 5% CO.sub.2
incubator to induce a transformant showing 50 nmol/l MTX
resistance. Binding activity of FGF8b-Fc fusion protein in the
supernatant in wells where growth of the transformant was observed,
to KM1334 was measured by the ELISA described in the item 4 of
Example 14.
[1016] Regarding the transformant in wells where production of
FGF-8/Fc fusion protein was observed in the culture supernatant,
according to the method similar to the above, a transformant KC1178
was obtained, which could grow in Hybridoma-SFM FBS(5) medium
comprising 0.5 mg/ml G418 and 200 nmol/l MTX as final
concentrations by increase of the MTX concentration and highly
produce FGF8-Fc son protein. It was found that KC1178 was a
lectin-resistant clone having a relatively low transcript amount
obtain ed by the measuring method of FUT8 gene transcript described
in Example 8 of WO00/61739. Also, KC1178 has been deposited on Apr.
1, 2003, as FERM BP-8350 in International Patent Organism
Depositary, National Institute of Advanced Industrial Science and
Technology (AIST Tsukuba Central 6, 1-1, Higashi 1-Chome
Tsukuba-shi, Ibaraki-ken, Japan).
[1017] (2) Preparation of Producing Cell Using CHO/DG44 Cells
[1018] After 10 .mu.g of the FGF8b-Fc fusion protein expression
vector pKANTEX/FGF8c was introduced into 1.6.times.10.sup.6 cells
of CHO/DG 44 cell by electroporation, the resulting cells were
suspended in 30 ml of IMDM-dFBS(10)-HT(1) and the solution was
dispensed at 100 .mu.l/well into a 96-well culture plate
(manufactured by Sumitomo Bakelite). After culturing at 37.degree.
C. for 24 hours in a 5% C.sub.2 incubator, the medium was changed
to IMDM-dFBE(10), followed by culturing for 1 to 2 weeks. The
culture supernatant was recovered from wells in which colonies of
transformants showing HT-independent growth were formed and growth
of colonies was observed, and the binding activity of the FGF8-Fc
fusion protein in the supernatant to KM1334 was measured by the
ELISA shown in the item 4 of Example 14.
[1019] Regarding the transformants in wells in which production of
the FGF-g/Fc protein was observed in culture supernatants, in order
to increase the production amount of the fusion protein by using a
dhfr gene amplification system, each of them was suspended in the
IMDM-dFBS(10) medium comprising 50 nmol/L of MTX (manufactured by
SIGMA), and dispensed into each well of a 24 well plate for
expansion culturing. After culturing at 37.degree. C. for 1 to 2
weeks in a 5% CO.sub.2 incubator to induce a transformant showing
50 nmol/l MTX resistance. Binding activity of FGF8b-Fc fusion
protein in the supernatant in wells where growth was observed, to
KM1334 was measured by the ELISA described in the item 4 of Example
14.
[1020] Regarding the transformant in wells where production of
FGF-8/Fc fusion protein was observed in the culture supernatant,
according to the method similar to the above, a transformant KC1179
was obtained, which could grow in IMDM-dFBS(10) medium comprising
500 nmol/l MTX by increase of the MTX concentration and highly
produce FGF8-Fc fusion protein. Also, KC11179 has been deposited on
Apr. 1, 2003, as FERM BP-8351 in International Patent Organism
Depositary, National Institute of Advanced Industrial Science and
Technology (Tsukuba Central 6, 1, Higashi 1-Chome Tsukuba-shi,
Ibaraki-ken, Japan).
[1021] 3. Purification of FGF8-Fc Fusion Protein
[1022] The producing cell of FGF8-Fc fusion protein prepared in the
item 2 of Example 14 was cultured in an appropriate culture medium
(e.g., H-SFM comprising 5% GF21 (manufactured by Wako Pure Chemical
Industries), 0.5 mg/ml of G418 and 200 mmol/l of MTX for YB2/0
cell-derived cells; EXCELL301 (manufactured by JRH) comprising 500
nmol/l MTX for CHO/D644 cell-derived cells) at a scale of 100 to
200 ml. FGF8-Fc fusion protein was purified from the culture
supernatant by using Prosep G (manufactured by Millipore) column
according to the manufacture's instruction. The deduced amino acid
sequence of the purified protein is shown by SEQ ID NO:90.
[1023] 4. Binding Activity to Anti-FGF-9 Antibody
[1024] Binding activities of the FGF-8/Fc fusion protein produced
by YB2/0 and the FGF-S/Fc fusion protein produced by CHO described
in the item 2 of Example 14, to KM1334 were measured by the ELISA
as follows. KM1334 of 1 .mu.g/ml was dispensed at 50 .mu.l/well
into a 96-well plate for ELISA (manufactured by Greiner) and
allowed to stand at 4.degree. C. overnight for adsorption After
washing with PBS, 1% BSA-PBS was added at 100 .mu.l/well and
allowed to react at room temperature for 1 hour to block the
remaining active group. After washing each well with Tween-PBS, the
culture supernatant of the transformant or a purified protein was
added at 50 .mu.l/well, followed reaction at room temperature.
After the reaction, each well was washed with Tween-PBS, and
peroxidase labeled goat-anti-human IgG(.gamma.) antibody solution
(manufactured by American Qualex), which was diluted 3000-fold with
1% BSA-PBS, was added at 50 .mu.l/well as the secondary antibody
solution, followed by reaction at room temperature for 1 hour.
After the reaction, each well was washed with Tween-PBS, and ABTS
substrate solution was added at 50 .mu.l/well, followed by
reaction. After the color developed sufficiently, 5% SDS solution
was added at 50 ed/well to stop the reaction. Then, the absorbance
was measured at a wavelength of 415 nm and a reference wavelength
of 490. As shown in FIG. 35, the FGF-8/Fc fusion protein obtained
in the item 2 of Example 14 showed binding activity to KM1334.
[1025] 5. Binding Activity to Fc.gamma.RIIIa
[1026] Binding activities of FGF-8/Fc fusion protein produced by
YB2/0 and FGF-8/Fc fusion protein derived from CHO described in the
item 2 of Example 14, to Fc.gamma.RIIa was measured by ELISA as
follows. Tetra-His Antibody, mouse antibody against is tag
(manufactured by QIAGEN), of 5 .mu.g/ml was dispensed at 50
.mu.l/well into a 96-well plate for ELISA (manufactured by GREINER)
and allowed to stand at 4.degree. C. overnight for adsorption.
After washing with PBS, 1% BSA-PBS was added at 100 .mu.l/well,
followed by reaction at room temperature for 1 hour to block the
remaining active group. After washing each well with Tween-PBS,
shFc.gamma.RIIIa (V) solution diluted to 5 .mu.g/ml with 1% BSA-PBS
was added at 50 .mu.l/well, followed by reaction at room
temperature for 2 hours. After the reaction, each well was washed
with Tween-PBS, and a solution prepared by diluting purified
FGF-8/Fc fusion protein to different concentrations with 1% BSA-PBS
was added at 50 .mu.l/well, followed by reaction at room
temperature for 2 hours. After the reaction, each well was washed
with Tween-PBS, and biotinized KM1334 diluted to 1 .mu.g/ml with 1%
BSA-PBS was added at 50 .mu.l/well, followed by reaction at room
temperature for 1 hour. After the reaction, each well was washed
with Tween-PBS, and a solution of peroxidase labeled Avidin-D
(manufactured by VECTOR) diluted 4000-fold with 1% BSA-PBS was
added at 50 .mu.l/well, followed by reaction at room temperature
for 1 hour. After the reaction, each well was washed with
Tween-PBS, and ABTS substrate solution was added at 50 .mu.l/well
for color development, and after 15 minutes, 5% SDS solution was
added at 50 .mu.l/well to stop the reaction. The absorbance of the
resulting solution was measured at a wavelength of 415 nm and a
reference wavelength of 490 nm.
[1027] FIG. 36 shows the measurement results of the binding
activity of various FGF-8/Fc fusion proteins to shFc.gamma.RIIIa
(V). As apparent in FIG. 36, the FGF-8/Fc fusion protein produced
by YB2/0 showed higher binding activity to shFc.gamma.RIIIa (V)
than the FGF-8/Fc fusion protein produced by CHO/DG44 cell.
REFERENCE EXAMPLE 1
[1028] Preparation of genes Encoding Various Enzymes Relating to
Sugar Chain Synthesis Derived from CHO Cell:
[1029] 1. Determination of FX cDNA Sequence in CHO Cell
[1030] (1) Extraction of Total RNA Derived from CHO/DG44 Cell
[1031] CHO/DG44 cells were suspended in IMDM medium containing
100/a fetal bovine serum (manufactured by Life Technologies) and
1.times. concentration HT supplement (manufactured by Life
Technologies), and 15 ml of the suspension was inoculated into a
T75 flask for adhesion cell culture use (manufactured by Greiner)
to give a density of 2.times.10 cells/ml. On the second day after
culturing at 37.degree. C. in a 5% CO.sub.2 incubator,
1.times.10.sup.7 of the cells were recovered and a total RNA was
extracted therefrom by using RNAeasy (manufactured by QIAGEN) in
accordance with the manufacture's instructions.
[1032] (2) Preparation of Total Single-Stranded cDNA from CHO/DG44
Cell
[1033] The total RNA prepared in the item 1(1) of Reference Example
1 was dissolved in 45 .mu.l of sterile water, and 1 .mu.l of RQ1
RNase-Free DNase (manufactured by Promega), 5 .mu.l of the attached
10.times. DNase buffer and 0.5 of RNasin Ribonuclease Inhibitor
(manufactured by Promega) were added thereto, followed by reaction
at 37.degree. C. for 30 minutes to degrade genomic DNA contaminated
in the sample. After the reaction, the total RNA was purified again
using RNAeasy (manufactured by QIAGEN) and dissolved in 50 .mu.l of
sterile water.
[1034] In a 20 .mu.l of reaction mixture using oligo(dT) as a
primer, single-stranded cDNA was synthesized from 3 .mu.g of the
obtained total RNA samples by carrying out reverse transcription
reaction using SUPERSCRIPT.TM. Preamplification System for First
Strand cDNA Synthesis (manufactured by Life Technologies) in
accordance with the manufacture's instructions. A 50 fold-diluted
aqueous solution of the reaction solution was used in the cloning
of GFPP and FX. This was stored at -80.degree. C. until use,
[1035] (3) Preparation Method of cDNA Partial Fragment of Chinese
Hamster-Derived FX
[1036] An FX cDNA partial fragment derived from Chinese hamster was
prepared by the following procedure.
[1037] First, primers (represented by SEQ ID NOs:42 and 43)
specific for common nucleotide sequences registered at a public
data base, namely a human FX cDNA (Genebank Accession No. U58766)
and a mouse cDNA (Genebank Accession No. M30127), were
designed.
[1038] Next, 25 .mu.l of a reaction solution [1.times.
concentration ExTaq buffer (manufactured by Takara Shuzo), 0.2
mmol/l dNTPs and 0.5 .mu.mol/l of the above gene-specific primers
(SEQ ID NOs:42 and 43)] containing 1 .mu.l of the CHO/DG44-derived
single-stranded cDNA prepared in the item 1(2) of Reference Example
1 was prepared by using a DNA polymerase ExTaq (manufactured by
Takara Shuzo), and PCR was carried out. The PCR was carried out by
heating at 94.degree. C. for 5 minutes, subsequent 30 cycles of
heating at 94.degree. C. for 1 minute, 58.degree. C. for 2 minutes
and 72.degree. C. for 3 minutes as one cycle, and further heating
at 72.degree. C. for 10 minutes.
[1039] After the PCR, the reaction solution was subjected to 2%
agarose gel electrophoresis, and a specific amplified fragment of
301 bp was purified using QuiaexII Get Extraction Kit (manufactured
by QIAGEN) and eluted with 20 .mu.l of sterile water (hereinafter,
the method was used for the purification of DNA fragments from
agarose gel). Into a plasmid pCR2.1, 4 .mu.l of the above amplified
fragment was employed to insert in accordance with the instructions
attached to TOPO TA Cloning Kit (manufactured by Invitrogen), and
E. coli DH5.alpha. was transformed with the reaction solution. Each
plasmid DNA was isolated in accordance with a known method from the
obtained several kanamycin-resistant colonies to obtain 2 clones
into which FX cDNA partial fragments were respectively inserted.
They were named pCRFX clone 8 and pCRFX clone 12.
[1040] The nucleotide sequence of the cDNA inserted into each of
the FX clone 8 and FX clone 12 was determined using DNA Sequencer
377 (manufactured by Parkin Elmer) and BigDye Terminator Cycle
Sequencing FS Ready Reaction kit (manufactured by Parkin Elmer) in
accordance with the method of the manufacture's instructions. It
was confirmed that each of the inserted cDNA whose sequence was
determined encodes an ORF partial sequence of the Chinese hamster
FX.
[1041] (4) Synthesis of Single-Stranded cDNA for RACE
[1042] Single-stranded cDNA samples for 5' and 3' RACE were
prepared from the CHO/DG44 total RNA extracted in the item 1(1) of
Reference Example 1 using SMART.TM. RACE cDNA Amplification Kit
(manufactured by CLONTECH) in accordance with the manufacture's
instructions. In the case, PowerScript.TM. Reverse Transcriptase
(manufactured by CLONTECH) was used as the reverse transcriptase.
Each single-stranded cDNA after the preparation was diluted 10-fold
with the Tricin-EDTA buffer attached to the kit and used as the
template of PCR.
[1043] (5) Determination of Chinese Hamster-Derived FX Full Length
cDNA by RACE Method
[1044] Based on the FX partial sequence derived from Chinese
hamster determined in the item 1(3) of Reference Example 1, primers
FXGSP1-1 (SEQ ID NO:44) and FXGSP1-2 (SEQ ID NO:45) for the Chinese
hamster FX-specific 5' RACE and primers FXGSP2-1 (SEQ DD NO:46) and
FXGSP2-2 (SEQ ED NO:47) for the Chinese hamster FX-specific 3' RACE
were designed.
[1045] Next, 50 .mu.l of a reaction solution [1.times.
concentration Advantage2 PCR buffer (manufactured by CLONTECH), 0.2
mmol/L dNTPs, 0.2 .mu.mol/l Chinese hamster FX-specific primers for
RACE and 1.times.concentration of common primers (manufactured by
CLONTECH)] containing 1 .mu.l of the CHO/DG44-derived
single-stranded cDNA for RACE prepared in the item 1(4) of
Reference Example 1 was prepared by using Advantage2 PCR Kit
(manufactured by CLONTECH) and PCR was carried out. The PCR was
carried out by 20 cycles of heating at 94.degree. C. for 5 seconds,
68.degree. C. for 10 seconds and 72.degree. C. for 2 minutes as one
cycle.
[1046] After completion of the reaction, 1 .mu.l of the reaction
solution was diluted 50-fold with the Tricin-EDTA buffer, and 1
.mu.l of the diluted solution was used as a template. The reaction
solution was again prepared and the PCR was carried out under the
same conditions. The templates, the combination of primers used in
the first and second PCRs and the length of amplified DNA fragments
by the PCRs are shown in Table 6.
6TABLE 6 Combination of primers used in Chinese hamster FX cDNA
RACE PCR and the size of PCR products FX-specific PCR-amplified
primers Common primers product size 5' RACE First FXGSP1-1 UPM
(Universal primer mix) Second FXGSP1-2 NUP (Nested Universal 300 bp
primer) 3' RACE First FXGSP2-1 UPM (Universal primer mix) Second
FXGSP2-2 NUP (Nested Universal 1,100 bp primer)
[1047] After the PCR, the reaction solution was subjected to 1%
agarose gel electrophoresis, and the specific amplified fragment of
interest was recovered and eluted with 20 .mu.l of sterile water.
Into a plasmid pCR2.1, 4 .mu.l of the amplified fragment was
inserted, and E. coli DH5.alpha. was transformed by using the
reaction solution in accordance with the instructions attached to
TOPO TA Cloning Kit (manufactured by Invitrogen). Plasmid DNAs were
isolated from the obtained kanamycin-resistant colonies to obtain 5
cDNA clones containing Chinese hamster FX 5' region. They were
named FX5' clone 25, FX5' clone 26, FX5' clone 27, FX5' clone 28,
FX5' clone 31 and FX5' clone 32.
[1048] In the same manner, 5 cDNA clones containing Chinese hamster
FX 3' region were obtained. These FX3' clones were named FX3' clone
1, FX3' clone 3, FX3' clone 6, FX3' clone 8 and FX3' clone 9.
[1049] The nucleotide sequence of the cDNA moiety of each of the
clones obtained by the 5' and 3' RACE was determined by using DNA
Sequencer 377 (manufactured by Parkin Elmer) in accordance with the
method described in the manufacture's instructions. By comparing
the cDNA nucleotide sequences determined by the method, reading
errors of nucleotide bases due to PCR were excluded and the full
length nucleotide sequence of Chinese hamster FX cDNA was
determined. The determined sequence is represented by SEQ ID
NO:48.
[1050] 2. Determination of CHO Cell-Derived GFPP cDNA Sequence
[1051] (1) Preparation of GFPP cDNA Partial Fragment Derived from
Chinese Hamster
[1052] GFPP cDNA partial fragment derived from Chinese hamster was
prepared by the following procedure.
[1053] First, nucleotide sequences of a human GFPP cDNA (Genebank
Accession No. AF017445), mouse EST sequences having high homology
with the sequence (Genebank Accession Nos. AI467195, AA422658,
BE304325 and AI466474) and rat EST sequences (Genebank Accession
Nos. BF546372, AI058400 and AW144783), registered at public data
bases, were compared, and primers GFPP FW9 and GFPP RV9. (SEQ ID
NOs:49 and 50) specific for rat GFPP were designed on a highly
preserved region among these three species.
[1054] Next, 25 .mu.l of a reaction solution [1.times.
concentration ExTaq buffer (manufactured by Takara Shuzo), 0.2
mmol/L dNTPs and 0.5 .mu.mol/l of the above GFPP-specific primers
GFPP FW9 and GFPP RV9 (SEQ ID NOs:49 and 50)] containing 1 .mu.l of
the CHO/DG44-derived single-stranded cDNA prepared in the item 1(2)
of Reference Example 1 was prepared by using a DNA polymerase ExTaq
(manufactured by Takara Shuzo), and PCR was carried out. The PCR
was carried out by heating at 94.degree. C. for 5 minutes,
subsequent 30 cycles of heating at 94.degree. C. for 1 minute,
58.degree. C. for 2 minutes and 72.degree. C. for 3 minutes as one
cycle, and further heating at 72.degree. C. for 10 minutes.
[1055] After the PCR, the reaction solution was subjected to 2%
agarose gel electrophoresis, and a specific amplified fragment of
1.4 Kbp was recovered and eluted with 20 .mu.l of sterile water.
Into a plasmid pCR2.1, 4 .mu.l of the above amplified fragment was
inserted in accordance with the instructions attached to TOPO TA
Cloning Kit (manufactured by Invitrogen), and E. coli DH5.alpha.
was transformed by using the reaction solution. Plasmid DNAs were
isolated from the obtained kanamycin resist ant clones to obtain 3
clones into which GFPP cDNA partial fragments were respectively
integrated. They were named GFPP clone 8, GFPP clone 11 and GFPP
clone 12.
[1056] The nucleotide sequence of the cDNA inserted into each of
the GFPP clone 8, GFPP clone 11 and GFPP clone 12 was determined by
using DNA Sequencer 377 (manufactured by Parkin Elmer) and BigDye
Terminator Cycle Sequencing FS Ready Reaction kit (manufactured by
Parkin Elmer) in accordance with the method described in the
manufacture's instructions. It was confirmed that the inserted cDNA
whose sequence was determined according to the present invention en
codes an ORF partial sequence of the Chinese hamster GFPP.
[1057] (2) Determination of Chinese Hamster GFPP Full Length cDNA
by RACE Method
[1058] Based on the Chinese hamster FX partial sequence determined
in the item 2(1) of Reference Example 1, primers GFPP GSP1-1 (SEQ
ID NO:52) and GFPP GSP1-2 (SEQ ID NO:53) for the Chinese hamster
FX-specific 5' RACE and primers GFPP GSP2-1 (SEQ ID NO:0.54) and
GFPP GSP2-2 (SEQ ID NO:55) for the Chinese hamster GFPP-specific 3,
RACE were designed.
[1059] Next, 50 .mu.l was of a reaction solution [1.times.
concentration Advantage2 PCR buffer (manufactured by CLONTECH), 0.2
mmol/L dNTPs, 0.2 .mu.mol/l Chinese hamster GFPP-specific primers
for RACE and 1.times. concentration of common primers (manufactured
by CLONTECH)] containing 1 .mu.l of the CHO/DG44 cell-derived
single-stranded cDNA for RACE prepared in the item 1(4) of
Reference Example 1 was prepared by using Advantage2 PCR Kit
(manufactured by CLONTECH), and PCR was carried out. The PCR was
carried out by 20 cycles of heating at 94.degree. C. for 5 seconds,
68.degree. C. for 10 seconds and 72.degree. C. for 2 minutes as one
cycle.
[1060] After completion of the reaction, 1 .mu.l of the reaction
solution was diluted 50-fold with the Tricin-EDTA buffer, and 1
.mu.l of the diluted solution was used as a template. The reaction
solution was again prepared and the PCR was carried out under the
same conditions. The templates, the combination of primers used in
the first and second PCRs and the size of amplified DNA fragments
by the PCRs are shown in Table 7.
7TABLE 7 Combination of primers used in Chinese hamster GFPP cDNA
RACE PCR and the size of PCR products GFPP-specific PCR-amplified
primers Common primers product size 5' RACE First GFPPGSP1-1 UPM
(Universal primer mix) Second GFPPGSP1-2 NUP (Nested Universal
1,100 bp primer) 3' RACE First GFPPGSP2-1 UPM (Universal primer
mix) Second GFPPGSP2-2 NUP (Nested Universal 1,400 bp primer)
[1061] After the PCR, the reaction solution was subjected to 1%
agarose gel electrophoresis, and the specific amplified fragment of
interest was recovered and eluted with 20 .mu.l of sterile water.
Into a plasmid pCR2.1, 4 .mu.l of the above amplified, fragment was
inserted and E. coli DH5.alpha. was transformed with the reaction
solution in accordance with the instructions attached to TOPO TA
Cloning Kit (manufactured by Invitrogen). Plasmid DNAs were
isolated from the obtained several kanamycin-resistant clones to
obtain 4 cDNA clones containing Chinese hamster GFPP 5' region.
They were named GFPP5' clone 1, GFPP5' clone 2, GFPP5' clone 3 and
GFPP5' clone 4.
[1062] In the same manner, 5 cDNA clones containing Chinese hamster
GFPP 3' region were obtained. They were named GFPP3' clone 10,
GFPP3' clone 16 and GFPP3' clone 20.
[1063] The nucleotide sequence of the cDNA protein of each of the
clones obtained in the above 5' and 3' RACE was determined by using
DNA Sequencer 377 (manufactured by Parkin Elmer) in accordance with
the method described in the manufacture's instructions. By
comparing the cDNA nucleotide sequences after the nucleotide
sequence determination, reading errors of bases due to PCR were
excluded and the full length nucleotide sequence of Chinese hamster
GFPP cDNA was determined. The determined sequence is represented by
SEQ ID NO:5.
REFERENCE EXAMPLE 2
[1064] Preparation of CHO Cell-Derived GMD Gene:
[1065] 1. Determination of CHO Cell-Derived GMD cDNA Sequence
[1066] (1) Preparation of CHO Cell-Derived GMD Gene cDNA
(Preparation of Partial cDNA Excluding 5'- and 3'-Terminal
Sequences)
[1067] Rodents-derived GMD cDNA was searched in a public data base
(BLAST) using a human-derived GMD cDNA sequence (GenBank Accession
No. AF042377) registered at GenBank as a query, and three kinds of
mouse EST sequences were obtained (GenBank Accession Nos. BE986856,
BF158988 and BE284785). By ligating these EST sequences, a deduced
mouse GMD cDNA sequence was determined.
[1068] On the basis of the mouse-derived GMD cDNA sequence, a 28
mer primer having the nucleotide sequence represented by SEQ ID
NO:56, a 27 mer primer having the nucleotide sequence represented
by SEQ ID NO:57, a 25 mer primer having the nucleotide sequence
represented by SEQ ID NO:58, a 24 mer primer having the nucleotide
sequence represented by SEQ ID NO:59 and a 25 mer primer having the
sequence represented by SEQ ID NO:60 were prepared.
[1069] Next, in order to amplify the CHO cell-derived GMD cDNA, PCR
was carried out by the following method. A reaction solution at 20
.mu.l [1.times. concentration Ex Taq buffer (manufactured by Takara
Shuzo), 0.2 mmol/L dNTPs, 0.5 unit of Ex Taq polymerase
(manufactured by Takara Shuzo) and 0.5 .mu.mol/L of two synthetic
DNA primers] containing 0.5 .mu.l of the CHO cell-derived
single-stranded cDNA prepared in the item 1(1) of Example 8 as the
template was prepared. In this case, combinations of SEQ ED NO:56
with SEQ ID NO:57, SEQ ID NO:58 with SEQ ID NO:57, SEQ ID NO:56
with SEQ ID NO:59 and SEQ ID NO:56 with SEQ ID NO:60 were used as
the synthetic DNA primers. The reaction was carried out by using
DNA Thermal Cycler 480 (manufactured by Perkin Elmer) by heating at
94.degree. C. for 5 minutes and subsequent 30 cycles of heating at
94.degree. C. for 1 minute and 68.degree. C. for 2 minutes as one
cycle.
[1070] The PCR reaction solution was fractionated by agarose
electrophoresis to find that a DNA fragment of about 1.2 kbp was
amplified in the PCR product when synthetic DNA primers of SEQ ID
NOs:56 and 57 were used, a fragment of about 1.1 kbp was amplified
in the PCR product when synthetic DNA primers of SEQ ID NOs:57 and
59 were used, a fragment of about 350 bp was amplified in the PCR
product when synthetic DNA primers of SEQ ID NOs:56 and 59 were
used and a fragment of about 1 kbp was amplified in the PCR product
when synthetic DNA primers of SEQ ID NOs:56 and 60 were used. The
DNA fragments were recovered. The recovered DNA fragments were
ligated to a pT7Blue(R) vector (manufactured by Novagen) by using
DNA Ligation Kit (manufactured by Takara Shuzo), and E. coli DH5
strain (manufactured by Toyobo) was transformed by using the
obtained recombinant plasmid DNA samples to thereby obtain plasmids
22-8 (having a DNA fragment of about 1.2 kbp amplified from
synthetic DNA primers of SEQ ID NO:56 and SEQ ID NO:57), 23-3
(having a DNA fragment of about 1.1 kbp amplified from synthetic
DNA primers of SEQ ID NO:58 and SEQ ID NO:57), 31-5 (a DNA fragment
of about 350 bp amplified from synthetic DNA primers of SEQ ID
NO:56 and SEQ ID NO:59) and 34-2 (having a DNA fragment of about 1
kbp amplified from synthetic DNA primers of SEQ ID NO:56 and SEQ ID
NO:60). The CHO cell-derived GMD cDNA sequence contained in these
plasmids was determined in the usual way by using a DNA sequencer
ABI PRISM 377 (manufactured by Parkin Elmer) (since a sequence of
28 bases in downstream of the initiation codon methionine in the
5'-terminal side and a sequence of 27 bases in upstream of the
termination codon in the 3'-terminal side are originated from
synthetic oligo DNA sequences, they are mouse GMD cDNA
sequences).
[1071] In addition, the following steps were carried out in order
to prepare a plasmid in which the CHO cell-derived GMD cDNAs
contained in the plasmids 22-8 and 34-2 are combined. The plasmid
22-8 (1 .mu.g) was allowed to react with a restriction enzyme EcoRI
(manufactured by Takara Shuzo) at 37.degree. C. for 16 hours, the
digest was subjected to agarose electrophoresis and then a DNA
fragment of about 4 kbp was recovered. The plasmid 34-2 (2 .mu.g)
was allowed to react with a restriction enzyme EcoRI at 37.degree.
C. for 16 hours, the digest was subjected to agarose
electrophoresis and then a DNA fragment of about 150 bp was
recovered. The recovered DNA fragments were respectively subjected
to terminal dephosphorylation using Calf Intestine Alkaline
Phosphatase (manufactured by Takara Shuzo) and then ligated by
using DNA Ligation Kit (manufactured by Takara Shuzo), and E. coli
DH5.alpha. strain (manufactured by Toyobo) was transformed by using
the obtained recombinant plasmid DNA to obtain a plasmid CHO-GMD
(cf. FIG. 37).
[1072] (2) Determination of 5'-Terminal Sequence of CHO
Cell-Derived GMD cDNA
[1073] A 24 mer primer having the nucleotide sequence represented
by SEQ ID NO:61 was prepared from 5'-terminal side non-coding
region nucleotide sequences of CHO cell-derived human and mouse GMD
cDNA, and a 32 mer primer having the nucleotide sequence
represented by SEQ ID NO:62 from CHO cell-derived GMD cDNA sequence
were prepared, and PCR was carried out by the following method to
amplify cDNA. Then 20 of a reaction solution [1.times.
concentration Ex Taq buffer (manufactured by Takara Shuzo), 0.2
mmol/L dNTPs, 0.5 unit of Ex Taq polymerase (manufactured by Takara
Shuzo) and 0.5 .mu.mol/L of the synthetic DNA primers of SEQ ID
NO:61 and SEQ ID NO:62] containing 0.5 .mu.l of the CHO
cell-derived single-stranded cDNA obtained in the item 1(1) of
Example 8 was prepared as the template, and the reaction was
carried out therein by using DNA Thermal Cycler 480 (manufactured
by Perkin Elmer) by heating at 94.degree. C. for 5 minutes,
subsequent 20 cycles of heating at 94.degree. C. for 1 minute,
55.degree. C. for 1 minute and 72.degree. C. for 2 minutes as one
cycle and further 18 cycles of heating at 94.degree. C. for 1
minute and 68.degree. C. for 2 minutes as one cycle. After
fractionation of the PCR reaction solution by agarose
electrophoresis, a DNA fragment of about 300 bp was recovered. The
recovered DNA fragment was ligated to a pT7Blue(R) vector
(manufactured by Novagen) by using DNA Ligation Kit (manufactured
by Takara Shuzo), and E. coli DH5.alpha. strain (manufactured by
Toyobo) was transformed by using the obtained recombinant plasmid
DNA samples to thereby obtain a plasmid 5'GMD. Using DNA Sequencer
377 (manufactured by Parkin Elmer), the nucleotide sequence of 28
bases in downstream of the initiation methionine of CHO-derived GMD
cDNA contained in the plasmid was determined.
[1074] (3) Determination of 3'-Terminal Sequence of CHO
Cell-Derived GMD cDNA
[1075] In order to obtain 3'-terminal cDNA sequence of CHO
cell-derived GMD, RACE method was carried out by the following
method. A single-stranded cDNA for 3' RACE was prepared from the
CHO cell-derived RNA obtained in the item 1(1) of Example 8 by
using SMART.TM. RACE cDNA Amplification Kit (manufactured by
CLONTECH) in accordance with the manufacture's instructions. In the
case, PowerScript.TM. Reverse Transcriptase (manufactured by
CLONTECH) was used as the reverse transcriptase. The
single-stranded cDNA after the preparation was diluted 10-fold with
the Tricin-EDTA buffer attached to the kit and used as the template
of PCR.
[1076] Next, 20 .mu.l of a react-on solution [1.times.
concentration ExTaq buffer (manufactured by Takara Shuzo), 0.2
mmol/L dNTPs, 0.5 unit of EX Taq polymerase (manufactured by Takara
Shuzo), 0.5 .mu.mol/L of the 24 mer synthetic DNA primer
represented by SEQ ID NO:63 [prepared on the basis of the CHO
cell-derived GMD cDNA sequence determined in the item 1(1) of
Reference Example 2] and 1.times. concentration of Universal Primer
Mix (attached to SMART.TM. RACE cDNA Amplification Kit;
manufactured by CLONTECH] containing 1 .mu.l of the above
single-stranded cDNA for 3, RACE as the template was prepared, and
the reaction was carried out by using DNA Thermal Cycler 480
(manufactured by Perkin Elmer) by heating at 94.degree. C. for 5
minutes and subsequent 30 cycles of heating at 94.degree. C. for 1
minute and 68.degree. C. for 2 minutes as one cycle.
[1077] After completion of the reaction, 1 .mu.l of the PCR
reaction solution was diluted 20-fold with Tricin-EDTA buffer
(manufactured by CLONTECH). Then, 20 .mu.l of a reaction solution
[1.times. concentration ExTaq buffer (manufactured by Takara
Shuzo), 0.2 mmol/L dNTPs, 0.5 unit of EX Taq polymerase
(manufactured by Takara Shuzo), 0.5 .mu.mol/L of the 25 mer
synthetic DNA primer represented by SEQ ID NO:64 [prepared on the
basis of the CHO cell-derived GMD cDNA sequence determined in the
item 1(1) of Reference Example 2] and 0.5 .mu.mol/L of Nested
Universal Primer (attached to SMART.TM. RACE cDNA Amplification
Kit; manufactured by CLONTECH)] containing 1 .mu.l of the 20
fold-diluted aqueous solution as the template was prepared, and the
reaction was carried out by using DNA Thermal Cycler 480
(manufactured by Perkin Elmer) by heating at 94.degree. C. for 5
minutes and subsequent 30 cycles of heating at 94.degree. C. for 1
minute and 68.degree. C. for 2 minutes as one cycle.
[1078] After completion of the reaction, the PCR reaction solution
was fractionated by agarose electrophoresis and then a DNA fragment
of about 700 bp was recovered. The recovered DNA fragment was
ligated to a pT7Blue(R) vector (manufactured by Novagen) by using
DNA Ligation Kit (manufactured by Takara Shuzo), and E. coli
DH5.alpha. strain (manufactured by Toyobo) was transformed by using
the obtained recombinant plasmid DNA, thereby obtaining a plasmid
3'GMD. Using DNA Sequencer 377 (manufactured by Parkin Elmer), the
nucleotide sequence of 27 bases in upstream of the termination
codon of CHO-derived GMD cDNA and 415 bp of 3'-terminal side
non-coding region contained in the plasmid was determined.
[1079] The full length cDNA sequence of the CHO-derived GMD gene
determined by the items 1(1), 1(2) and 1(3) of Reference Example 2
is represented by SEQ ID NO:65, and the corresponding amino acid
sequence is represented by SEQ ID NO:71.
[1080] 2. Determination of Genomic Sequence Containing
CHO/DG44-Derived Cell GMD Gene
[1081] A 25 mer primer having the nucleotide sequence represented
by SEQ ID NO:66 was prepared from the mouse GMD cDNA sequence
determined in the item 1 of Reference Example 2. Next, a CHO
cell-derived genomic DNA was obtained by the following method. A
CHO/DG44 cell-derived KC861 cell was suspended in
IMDM-dFBS(10)-HT(1) medium [IMDM-dFBS(10) medium comprising
1.times. concentration of HT supplement (manufactured by
Invitrogen)] to give a density of 3.times.10 cells/ml, and the
suspension was dispensed at 2 ml/well into a 6 well flat bottom
plate for adhesion cell use (manufactured by Greiner). After
culturing at 37.degree. C. in a 5% CO.sub.2 incubator until the
cells became confluent on the plate, genomic DNA was prepared from
the cells on the plate by a known method [Nucleic Acids Research,
3, 2303 (1976)] and dissolved overnight in 150 .mu.l of TE-RNase
buffer (pH 8.0) (10 mmol/l Tris-HCl, 1 mmol/l EDTA, 200 .mu.g/ml
RNase A).
[1082] A reaction solution (20 .mu.l) [1.times. concentration Ex
Taq buffer (manufactured by Takara Shuzo), 0.2 mmol/L dNTPs, 0.5
unit of EX Taq polymerase (manufactured by Takara Shuzo) and 0.5
.mu.mol/L of synthetic DNA primers of SEQ ID NO:59 and SEQ ID
NO:66] containing 100 ng of the obtained CHO/DG44 cell-derived
genomic DNA was prepared, and PCR was carried out by using DNA
Thermal Cycler 480 (manufactured by Perkin Elmer) by heating at
94.degree. C. for 5 minutes and subsequent 30 cycles of heating at
94.degree. C. for 1 minute and 68.degree. C. for 2 minutes as one
cycle. After completion of the reaction, the PCR reaction solution
was fractionated by agarose electrophoresis and then a DNA fragment
of about 100 bp was recovered. The recovered DNA fragment was
ligated to a pT7Blue(R) vector (manufactured by Novagen) by using
DNA Ligation Kit (manufactured by Takara Shuzo), and E. coli
DH5.alpha. strain (manufactured by Toyobo) was transformed by using
the obtained recombinant plasmid DNA, thereby obtaining a plasmid
ex3. Using DNA Sequencer 377 (manufactured by Parkin Elmer), the
nucleotide sequence of CHO cell-derived genomic DNA contained in
the plasmid was determined. The nucleotide sequence was represented
by SEQ ID NO:67.
[1083] Next, a 25 mer primer having the nucleotide sequence
represented by SEQ ID NO:68 and a 25 mer primer having the
nucleotide sequence represented by SEQ ID NO:69 were prepared on
the basis of the CHO cell-derived GMD cDNA sequence determined in
the item 1 of Reference Example 2. Next, 20 .mu.l of a reaction
solution [1.times. concentration Ex Taq buffer (manufactured by
Takara Shuzo), 0.2 mmol/L dNTPs, 0-5 unit of EX Taq polymerase
(manufactured by Takara Shuzo) and 0.5 mmol/L of the synthetic DNA
primers of SEQ ID NO:68 and SEQ ID NO:69] containing 100 ng of the
CHO/DG44-derived genomic DNA was prepared, and PCR was carried out
by using DNA Thermal Cycler 480 (manufactured by Perkin Elmer) by
heating at 94.degree. C. for 5 minutes and subsequent 30 cycles of
heating at 94.degree. C. for 1 minute and 68.degree. C. for 2
minutes as one cycle.
[1084] After completion of the reaction, the PCR reaction solution
was fractionated by agarose electrophoresis and then a DNA fragment
of about 200 bp was recovered. The recovered DNA fragment was
ligated to a pT7Blue(R) vector (manufactured by Novagen) by using
DNA Ligation Kit (manufactured by Takara Shuzo), and E. coli
DH5.alpha. strain (manufactured by Toyobo) was transformed by using
the obtained recombinant plasmid DNA, thereby obtaining a plasmid
ex4. Using DNA Sequencer 377 (manufactured by Parkin Elmer), the
nucleotide sequence of CHO cell-derived genomic DNA contained in
the plasmid was determined. The result nucleotide sequence was
represented by SEQ ID NO:70.
REFERENCE EXAMPLE 3
[1085] Preparation of Anti-FGF-8 Chimeric Antibody
[1086] 1. Isolation and Analysis of a cDNA Encoding the V Region of
a Mouse Antibody Against FGF-8
[1087] (1) Preparation of mRNA from Hybridoma Cells which Produces
a Mouse Antibody Against FGF-8.
[1088] About 8 .mu.g of mRNA was prepared from 1.times.10.sup.7
cells of a hybridoma KM1334 (FERM BP-5451) which produces a mouse
antibody against FGF-8 (anti-FGF-8 mouse antibody), using a mRNA
preparation kit Fast Track mRNA Isolation Kit (manufactured by
Invitrogen) according to the attached manufacture's
instructions.
[1089] (2) Production of cDNA Libraries of Anti-FGF-8 Mouse
Antibody H Chain and L Chain
[1090] A cDNA having EcoRI-NotI adapters on both termini was
synthesized from 5 .mu.g of the KM1334 mRNA obtained in the item
1(1) of Reference Example 3 by using Time Saver cDNA Synthesis Kit
(manufactured by Amersham Pharmacia Biotech) according to the
attached manufacture's instructions. A full amount of the prepared
cDNA was dissolved in 20 .mu.l of sterile water and then
fractionated by agarose gel electrophoresis, and about 1.5 kb of a
cDNA fragment corresponding to the H chain of an IgG class antibody
and about 1.0 kb of a cDNA fragment corresponding to the L chain of
a .kappa. class were recovered each at about 0.1 .mu.g. Next, 0.1
.mu.g of the cDNA fragment of about 1.5 kb and 0.1 .mu.g of the
cDNA fragment of about 1.0 kb were respectively digested with
restriction enzyme EcoRI and then ligated with 1 .mu.g of
.lambda.ZAPII vector whose termini had been dephosphorylated with
calf intestine alkaline phosphatase, using .lambda.ZAPII Cloning
Kit (manufactured by Stratagene) according to the attached
manufacture's instructions.
[1091] Using Gigapack II Packaging Extracts Gold (manufactured by
Stratagene), 4 .mu.l of each reaction solution after ligation was
packaged in .lambda. phage according to the manufacture's
instructions, and Escherichia coli XL1-Blue [Biotechniques, 5, 376
(1987)] was infected with an adequate amount of the package to
obtain about 8.1.times.10.sup.4 and 5.5.times.10.sup.4 phage clones
as H chain cDNA library and L chain cDNA library, respectively, of
KM334. Next, respective phages were immobilized on a nylon membrane
according to a known method (Molecular Cloning, Second
Edition).
[1092] (3) Cloning of cDNAs of Anti-FGF-8 Mouse Antibody H Chain
and L Chain
[1093] Nylon membranes of the H chain cDNA library and L chain cDNA
library of KM1334 prepared in the item 1(2) in Reference Example 3
were detected using a cDNA of the C region of a mouse antibody [H
chain is a DNA fragment containing mouse C.gamma.1 cDNA (J.
Immunol., 146, 2010 (1991)], L chain is a DNA fragment containing
mouse CK cDNA [Cell, 22, 197 (1980)] as a probe, using ECL Direct
Nucleic Acid Labeling and Detection Systems (manufactured by
Amersham Pharmacia Biotech) according to the attached manufacture's
instructions, and phage clones strongly linked to the probe, 10
clones for each of H chain and L chain, were obtained. Next, each
phage clone was converted into a plasmid by the in vivo excision
method according to the manufacture's instructions attached to
.lambda.ZAPII Cloning Kit (manufactured by Stratagene) A nucleotide
sequence of a cDNA contained in each of the obtained plasmids was
determined by the dideoxy method (Molecular Cloning, Second
Edition) by using Big Dye Terminator Kit ver. 2 (manufactured by
Applied Biosystems). As a result, a plasmid pKM1334H7-1 containing
a full length and functional H chain cDNA and a plasmid pKM1334L7-1
containing L chain cDNA, having an ATG sequence considered to be
the initiation codon in the 5'-terminal of the cDNA were
obtained.
[1094] (4) Analysis of Amino Acid Sequence of V Region of
Anti-FGF-8 Mouse Antibody
[1095] A full length nucleotide sequence of VH contained in the
plasmid pKM1334H7-1 and a deduced complete length amino acid
sequence are represented by SEQ ID NO:72 and SEQ ID NO:73,
respectively, and a full length nucleotide sequence of VL contained
in the plasmid pKM1334L7-1 and a deduced complete length amino acid
sequence are represented by SEQ ID NO:74 and SEQ ID NO:75,
respectively. As a result of comparing these sequences to both
known sequence data of mouse antibodies (Sequences of Proteins of
Immunological Interest, U.S. Dept. Health and Human Services, 1991)
and the comparison with the results of analysis of N-terminal amino
acid sequences of H chain and L chain of the purified anti-FGF-8
mouse antibody KM1334, carried out by their automatic Edman
degradation using a protein sequencer PPSQ-10 (manufactured by
Shimadzu), it was found that each of the isolated cDNA is a full
length cDNA encoding the anti-FGF-8 mouse antibody KM1334
containing a secretory signal sequence, and positions 1 to 19 in
the amino acid sequence represented by SEQ ID NO:73 and positions 1
to 19 in the amino acid sequence described in SEQ ID NO:75 are
secretory signal sequences of H chain and L chain,
respectively.
[1096] Next, novelty of the amino acid sequences (sequences
excluding secretory signal sequence) of VH and VL of the anti-FGF-8
mouse antibody KM1334 was examined. Using GCG Package (version 9.1
manufactured by Genetics Computer Group) as a sequence analyzing
system, an amino acid sequence data base of known proteins
(PIR-Protein (Release 56.0)) was searched by the BLAST method [J.
Mol. Biol., 215, 403 (1990)]. As a result, completely coincided
sequences were not found for both of the H chain and L chain, so
that it was confirmed that the VH and VL of the anti-FGF-8 mouse
antibody KM1334 are novel amino acid sequences.
[1097] Also, the CDR of VH and VL of the anti-FGF-8 mouse antibody
KM1334 was identified by comparing with amino acid sequences of
known antibodies. Amino acid sequences of CDR 1, 2 and 3 of VH of
the anti-FGF-8 mouse antibody KM1334 are represented by SEQ DD
NOs:76, 77 and 78, respectively, and amino acid sequences of CDR 1,
2 and 3 of VL in SEQ ID NOs:79, 80 and 81, respectively.
[1098] 2. Stable Expression of Anti-FGF-8 Chimeric Antibody Using
Animal Cell
[1099] (1) Construction of Anti-FGF-8 Chimeric Antibody Expression
Vector pKANTEX1334
[1100] An anti-FGF-8 chimeric antibody expression vector
pKANTEX1334 was constructed as follows using the vector pKANTEX93
for humanized antibody expression described in WO97/10354 and the
plasmids pKM1334H7-1 and pKM1334L7-1 obtained in the item 1(3) of
Reference Example 3.
[1101] Using 50 ng of the plasmid pKM1334H7-1 obtained in the item
1(3) of Reference Example 3 as the template and by adding synthetic
DNAs having the nucleotide sequences described in SEQ ID NOs:24 and
25 (manufactured by GENSET) as primers to give a final
concentration 0.3 .mu.M, PCR were carried out in a system of 50
.mu.l by first heating at 94.degree. C. for 2 minutes and
subsequent 30 cycles of heat at 94.degree. C. for 15 seconds, at
55.degree. C. for 30 seconds and at 68.degree. C. for 1 minute
according to the manufacture's instructions attached to KOD plus
polymerase (manufactured by TOYOBO). The reaction solution was
precipitated with ethanol, dissolved in sterile water and then
allowed to react at 37.degree. C. for 1 hour by using 10 units of a
restriction enzyme ApaI (manufactured by Takara Shuzo) and 10 units
of a restriction enzyme NotI (manufactured by New England Biolabs).
About 0.3 .mu.g of an ApaI-NotI fragment of about 0.47 kb was
recovered By fractionating the reaction solution by agarose gel
electrophoresis.
[1102] Next, 3 .mu.g of the vector pKANTEX93 for humanized antibody
expression was allowed to react at 37.degree. C. for 1 hour by
using 10 units of restriction enzyme ApaI (manufactured by Takara
Shuzo) and 10 units of restriction enzyme NotI (manufactured by New
England Biolabs). About 2 g of an ApaI-NotI fragment of about 12.75
kb was recovered, by fractionating the reaction solution by an
agarose gel electrophoresis.
[1103] Next, 0.1 .mu.g of the NotI-ApaI fragment derived from the
PCR product and 0.1 .mu.g of the NotI-ApaI fragment derived from
the plasmid pKANTEX93, obtained in the above, were added to 10
.mu.l of sterile water in total amount and ligated by using
Ligation High (manufactured by TOYOBO). The plasmid pKANTEX1334H
shown in FIG. 38 was obtained by transforming Escherichia coli
JM109 by using the recombinant plasmid DNA solution obtained in
this manner.
[1104] Next, using 50 ng of the plasmid pKM1334L7-1 obtained in the
item 1(3) of Reference Example 1 as the template and by adding
synthetic DNAs having the nucleotide sequences described in SEQ ID
NOs:82 and 83 (manufactured by GENSET) as primers to give a final
concentration of 0.3 .mu.M, PCR was carried out in a system of 50
.mu.l by first heating at 94.degree. C. for 2 minutes and
subsequent 30 cycles of heating at 94.degree. C. for 15 seconds, at
55.degree. C. for 30 seconds and 68.degree. C. for 1 minute
according to the manufacture's instructions attached to KOD plus
polymerase (manufactured by TOYOBO). The reaction solution was
precipitated with ethanol, dissolved in sterile water and then
allowed to react at 37.degree. C. for 1 hour by using 10 units of a
restriction enzyme EcoRI (manufactured by Takara Shuzo) and 10
units of a restriction enzyme BsiWI (manufactured by New England
Biolabs). About 0.3 .mu.g of an EcoRI-BsiWI fragment of about 0.44
kb was recovered by fractionating the reaction solution by agarose
gel electrophoresis.
[1105] Next, 3 .mu.g of the plasmid pKANTEX1134H obtained in the
above was allowed to react at 37.degree. C. for 1 hour by using 10
units of a restriction enzyme EcoRI (manufactured by Takara Shuzo)
and a restriction enzyme BsiWI (manufactured by New England
Biolabs). About 2 .mu.g of an EcoRI-BsiWI fragment of about 13.20
kb was recovered by fractionating said reaction solution by an
agarose gel electrophoresis.
[1106] Next, 0.1 .mu.g of the EcoRI-BsiWI fragment derived from the
PCR product and 0.1 .mu.g of the EcoRI-BsiWI fragment derived from
the plasmid pKANTEX1334H, obtained in the above, were added to 10
.mu.l of sterile water in total amount and ligated by using
Ligation High (manufactured by TOYOBO). The plasmid pKANTEX1334
shown in FIG. 38 was obtained by transforming Escherichia coli
JM109 using the recombinant plasmid DNA solution obtained in this
manner.
[1107] As a result of carrying out analysis of a nucleotide
sequence using 400 ng of the obtained plasmid by the dideoxy method
(Molecular Cloning, Second Edition) using Big Dye Terminator Kit
ver. 2 (manufactured by Applied Biosystems), it was confirmed that
a plasmid comprising a cloned DNA of interest was obtained.
[1108] While the invention has been described in detail and with
reference to specific embodiments thereof, it will be apparent to
one skill in the art that various changes and modifications can be
made therein without departing from the spirit and scope thereof.
All references cited herein are incorporated in their entirety.
[1109] This application is based on Japanese application No.
2002-106950 filed on Apr. 9, 2002, the entire contents of which
being incorporated hereinto by reference.
Sequence CWU 1
1
100 1 2008 DNA Cricetulus griseus 1 aacagaaact tattttcctg
tgtggctaac tagaaccaga gtacaatgtt tccaattctt 60 tgagctccga
gaagacagaa gggagttgaa actctgaaaa tgcgggcatg gactggttcc 120
tggcgttgga ttatgctcat tctttttgcc tgggggacct tattgtttta tataggtggt
180 catttggttc gagataatga ccaccctgac cattctagca gagaactctc
caagattctt 240 gcaaagctgg agcgcttaaa acaacaaaat gaagacttga
ggagaatggc tgagtctctc 300 cgaataccag aaggccctat tgatcagggg
acagctacag gaagagtccg tgttttagaa 360 gaacagcttg ttaaggccaa
agaacagatt gaaaattaca agaaacaagc taggaatgat 420 ctgggaaagg
atcatgaaat cttaaggagg aggattgaaa atggagctaa agagctctgg 480
ttttttctac aaagtgaatt gaagaaatta aagaaattag aaggaaacga actccaaaga
540 catgcagatg aaattctttt ggatttagga catcatgaaa ggtctatcat
gacagatcta 600 tactacctca gtcaaacaga tggagcaggt gagtggcggg
aaaaagaagc caaagatctg 660 acagagctgg tccagcggag aataacatat
ctgcagaatc ccaaggactg cagcaaagcc 720 agaaagctgg tatgtaatat
caacaaaggc tgtggctatg gatgtcaact ccatcatgtg 780 gtttactgct
tcatgattgc ttatggcacc cagcgaacac tcatcttgga atctcagaat 840
tggcgctatg ctactggagg atgggagact gtgtttagac ctgtaagtga gacatgcaca
900 gacaggtctg gcctctccac tggacactgg tcaggtgaag tgaaggacaa
aaatgttcaa 960 gtggtcgagc tccccattgt agacagcctc catcctcgtc
ctccttactt acccttggct 1020 gtaccagaag accttgcaga tcgactcctg
agagtccatg gtgatcctgc agtgtggtgg 1080 gtatcccagt ttgtcaaata
cttgatccgt ccacaacctt ggctggaaag ggaaatagaa 1140 gaaaccacca
agaagcttgg cttcaaacat ccagttattg gagtccatgt cagacgcact 1200
gacaaagtgg gaacagaagc agccttccat cccattgagg aatacatggt acacgttgaa
1260 gaacattttc agcttctcga acgcagaatg aaagtggata aaaaaagagt
gtatctggcc 1320 actgatgacc cttctttgtt aaaggaggca aagacaaagt
actccaatta tgaatttatt 1380 agtgataact ctatttcttg gtcagctgga
ctacacaacc gatacacaga aaattcactt 1440 cggggcgtga tcctggatat
acactttctc tcccaggctg acttccttgt gtgtactttt 1500 tcatcccagg
tctgtagggt tgcttatgaa atcatgcaaa cactgcatcc tgatgcctct 1560
gcaaacttcc attctttaga tgacatctac tattttggag gccaaaatgc ccacaaccag
1620 attgcagttt atcctcacca acctcgaact aaagaggaaa tccccatgga
acctggagat 1680 atcattggtg tggctggaaa ccattggaat ggttactcta
aaggtgtcaa cagaaaacta 1740 ggaaaaacag gcctgtaccc ttcctacaaa
gtccgagaga agatagaaac agtcaaatac 1800 cctacatatc ctgaagctga
aaaatagaga tggagtgtaa gagattaaca acagaattta 1860 gttcagacca
tctcagccaa gcagaagacc cagactaaca tatggttcat tgacagacat 1920
gctccgcacc aagagcaagt gggaaccctc agatgctgca ctggtggaac gcctctttgt
1980 gaagggctgc tgtgccctca agcccatg 2008 2 1728 DNA Mus musculus 2
atgcgggcat ggactggttc ctggcgttgg attatgctca ttctttttgc ctgggggacc
60 ttgttatttt atataggtgg tcatttggtt cgagataatg accaccctga
tcactccagc 120 agagaactct ccaagattct tgcaaagctt gaacgcttaa
aacagcaaaa tgaagacttg 180 aggcgaatgg ctgagtctct ccgaatacca
gaaggcccca ttgaccaggg gacagctaca 240 ggaagagtcc gtgttttaga
agaacagctt gttaaggcca aagaacagat tgaaaattac 300 aagaaacaag
ctagaaatgg tctggggaag gatcatgaaa tcttaagaag gaggattgaa 360
aatggagcta aagagctctg gttttttcta caaagcgaac tgaagaaatt aaagcattta
420 gaaggaaatg aactccaaag acatgcagat gaaattcttt tggatttagg
acaccatgaa 480 aggtctatca tgacagatct atactacctc agtcaaacag
atggagcagg ggattggcgt 540 gaaaaagagg ccaaagatct gacagagctg
gtccagcgga gaataacata tctccagaat 600 cctaaggact gcagcaaagc
caggaagctg gtgtgtaaca tcaataaagg ctgtggctat 660 ggttgtcaac
tccatcacgt ggtctactgt ttcatgattg cttatggcac ccagcgaaca 720
ctcatcttgg aatctcagaa ttggcgctat gctactggtg gatgggagac tgtgtttaga
780 cctgtaagtg agacatgtac agacagatct ggcctctcca ctggacactg
gtcaggtgaa 840 gtaaatgaca aaaacattca agtggtcgag ctccccattg
tagacagcct ccatcctcgg 900 cctccttact taccactggc tgttccagaa
gaccttgcag accgactcct aagagtccat 960 ggtgaccctg cagtgtggtg
ggtgtcccag tttgtcaaat acttgattcg tccacaacct 1020 tggctggaaa
aggaaataga agaagccacc aagaagcttg gcttcaaaca tccagttatt 1080
ggagtccatg tcagacgcac agacaaagtg ggaacagaag cagccttcca ccccatcgag
1140 gagtacatgg tacacgttga agaacatttt cagcttctcg cacgcagaat
gcaagtggat 1200 aaaaaaagag tatatctggc tactgatgat cctactttgt
taaaggaggc aaagacaaag 1260 tactccaatt atgaatttat tagtgataac
tctatttctt ggtcagctgg actacacaat 1320 cggtacacag aaaattcact
tcggggtgtg atcctggata tacactttct ctcacaggct 1380 gactttctag
tgtgtacttt ttcatcccag gtctgtcggg ttgcttatga aatcatgcaa 1440
accctgcatc ctgatgcctc tgcgaacttc cattctttgg atgacatcta ctattttgga
1500 ggccaaaatg cccacaatca gattgctgtt tatcctcaca aacctcgaac
tgaagaggaa 1560 attccaatgg aacctggaga tatcattggt gtggctggaa
accattggga tggttattct 1620 aaaggtatca acagaaaact tggaaaaaca
ggcttatatc cctcctacaa agtccgagag 1680 aagatagaaa cagtcaagta
tcccacatat cctgaagctg aaaaatag 1728 3 9196 DNA Cricetulus griseus 3
tctagaccag gctggtctcg aactcacaga gaaccacctg cctctgccac ctgagtgctg
60 ggattaaagg tgtgcaccac caccgcccgg cgtaaaatca tatttttgaa
tattgtgata 120 atttacatta taattgtaag taaaaatttt cagcctattt
tgttatacat ttttgcgtaa 180 attattcttt tttgaaagtt ttgttgtcca
taatagtcta gggaaacata aagttataat 240 ttttgtctat gtatttgcat
atatatctat ttaatctcct aatgtccagg aaataaatag 300 ggtatgtaat
agcttcaaca tgtggtatga tagaattttt cagtgctata taagttgtta 360
cagcaaagtg ttattaattc atatgtccat atttcaattt tttatgaatt attaaattga
420 atccttaagc tgccagaact agaattttat tttaatcagg aagccccaaa
tctgttcatt 480 ctttctatat atgtggaaag gtaggcctca ctaactgatt
cttcacctgt tttagaacat 540 ggtccaagaa tggagttatg taaggggaat
tacaagtgtg agaaaactcc tagaaaacaa 600 gatgagtctt gtgaccttag
tttctttaaa aacacaaaat tcttggaatg tgttttcatg 660 ttcctcccag
gtggatagga gtgagtttat ttcagattat ttattacaac tggctgttgt 720
tacttgtttc tatgtcttta tagaaaaaca tatttttttt gccacatgca gcttgtcctt
780 atgattttat acttgtgtga ctcttaactc tcagagtata aattgtctga
tgctatgaat 840 aaagttggct attgtatgag acttcagccc acttcaatta
ttggcttcat tctctcagat 900 cccaccacct ccagagtggt aaacaacttg
aaccattaaa cagactttag tctttatttg 960 aatgatagat ggggatatca
gatttatagg cacagggttt tgagaaaggg agaaggtaaa 1020 cagtagagtt
taacaacaac aaaaagtata ctttgtaaac gtaaaactat ttattaaagt 1080
agtagacaag acattaaata ttccttggga ttagtgcttt ttgaattttg ctttcaaata
1140 atagtcagtg agtatacccc tcccccattc tatattttag cagaaatcag
aataaatggt 1200 gtttctggta cattcttttg tagagaattt attttctttg
ggtttttgtg catttaaagt 1260 caataaaaat taaggttcag taatagaaaa
aaaactctga tttttggaat cccctttctt 1320 cagcttttct atttaatctc
ttaatgataa tttaatttgt ggccatgtgg tcaaagtata 1380 tagccttgta
tatgtaaatg ttttaaccaa cctgccttta cagtaactat ataattttat 1440
tctataatat atgacttttc ttccatagct ttagagttgc ccagtcactt taagttacat
1500 tttcatatat gttctttgtg ggaggagata attttatttc taagagaatc
ctaagcatac 1560 tgattgagaa atggcaaaca aaacacataa ttaaagctga
taaagaacga acatttggag 1620 tttaaaatac atagccaccc taagggttta
actgttgtta gccttctttt ggaattttta 1680 ttagttcata tagaaaaatg
gattttatcg tgacatttcc atatatgtat ataatatatt 1740 tacatcatat
ccacctgtaa ttattagtgt ttttaaatat atttgaaaaa ataatggtct 1800
ggtttgatcc atttgaacct tttgatgttt ggtgtggttg ccaattggtt gatggttatg
1860 ataacctttg cttctctaag gttcaagtca gtttgagaat atgtcctcta
aaaatgacag 1920 gttgcaagtt aagtagtgag atgacagcga gatggagtga
tgagaatttg tagaaatgaa 1980 ttcacttata ctgagaactt gttttgcttt
tagataatga acatattagc ctgaagtaca 2040 tagccgaatt gattaattat
tcaaagatat aatcttttaa tccctataaa agaggtatta 2100 cacaacaatt
caagaaagat agaattagac ttccagtatt ggagtgaacc atttgttatc 2160
aggtagaacc ctaacgtgtg tggttgactt aaagtgttta ctttttacct gatactgggt
2220 agctaattgt ctttcagcct cctggccaaa gataccatga aagtcaactt
acgttgtatt 2280 ctatatctca aacaactcag ggtgtttctt actctttcca
cagcatgtag agcccaggaa 2340 gcacaggaca agaaagctgc ctccttgtat
caccaggaag atctttttgt aagagtcatc 2400 acagtatacc agagagacta
attttgtctg aagcatcatg tgttgaaaca acagaaactt 2460 attttcctgt
gtggctaact agaaccagag tacaatgttt ccaattcttt gagctccgag 2520
aagacagaag ggagttgaaa ctctgaaaat gcgggcatgg actggttcct ggcgttggat
2580 tatgctcatt ctttttgcct gggggacctt attgttttat ataggtggtc
atttggttcg 2640 agataatgac caccctgacc attctagcag agaactctcc
aagattcttg caaagctgga 2700 gcgcttaaaa caacaaaatg aagacttgag
gagaatggct gagtctctcc ggtaggtttg 2760 aaatactcaa ggatttgatg
aaatactgtg cttgaccttt aggtataggg tctcagtctg 2820 ctgttgaaaa
atataatttc tacaaaccgt ctttgtaaaa ttttaagtat tgtagcagac 2880
tttttaaaag tcagtgatac atctatatag tcaatatagg tttacatagt tgcaatctta
2940 ttttgcatat gaatcagtat atagaagcag tggcatttat atgcttatgt
tgcatttaca 3000 attatgttta gacgaacaca aactttatgt gatttggatt
agtgctcatt aaattttttt 3060 attctatgga ctacaacaga gacataaatt
ttgaaaggct tagttactct taaattctta 3120 tgatgaaaag caaaaattca
ttgttaaata gaacagtgca tccggaatgt gggtaattat 3180 tgccatattt
ctagtctact aaaaattgtg gcataactgt tcaaagtcat cagttgtttg 3240
gaaagccaaa gtctgattta aatggaaaac ataaacaatg atatctattt ctagatacct
3300 ttaacttgca gttactgagt ttacaagttg tctgacaact ttggattctc
ttacttcata 3360 tctaagaatg atcatgtgta cagtgcttac tgtcacttta
aaaaactgca gggctagaca 3420 tgcagatatg aagactttga cattagatgt
ggtaattggc actaccagca agtggtatta 3480 agatacagct gaatatatta
ctttttgagg aacataattc atgaatggaa agtggagcat 3540 tagagaggat
gccttctggc tctcccacac cactgtttgc atccattgca tttcacactg 3600
cttttagaac tcagatgttt catatggtat attgtgtaac tcaccatcag ttttatcttt
3660 aaatgtctat ggatgataat gttgtatgtt aacactttta caaaaacaaa
tgaagccata 3720 tcctcggtgt gagttgtgat ggtggtaatt gtcacaatag
gattattcag caaggaacta 3780 agtcagggac aagaagtggg cgatactttg
ttggattaaa tcattttact ggaagttcat 3840 cagggagggt tatgaaagtt
gtggtctttg aactgaaatt atatgtgatt cattattctt 3900 gatttaggcc
ttgctaatag taactatcat ttattgggaa tttgtcatat gtgccaattt 3960
gtcatgggcc agacagcgtg ttttactgaa tttctagata tctttatgag attctagtac
4020 tgttttcagc cattttacag atgaagaatc ttaaaaaatg ttaaataatt
tagtttgccc 4080 aagattatac gttaacaaat ggtagaacct tctttgaatt
ctggcagtat ggctacacag 4140 tccgaactct tatcttccta agctgaaaac
agaaaaagca atgacccaga aaattttatt 4200 taaaagtctc aggagagact
tcccatcctg agaagatctc ttttcccttt tataatttag 4260 gctcctgaat
aatcactgaa ttttctccat gttccatcta tagtactgtt atttctgttt 4320
tccttttttc ttaccacaaa gtatcttgtt tttgctgtat gaaagaaaat gtgttattgt
4380 aatgtgaaat tctctgtccc tgcagggtcc cacatccgcc tcaatcccaa
ataaacacac 4440 agaggctgta ttaattatga aactgttggt cagttggcta
gggcttctta ttggctagct 4500 ctgtcttaat tattaaacca taactactat
tgtaagtatt tccatgtggt cttatcttac 4560 caaggaaagg gtccagggac
ctcttactcc tctggcgtgt tggcagtgaa gaggagagag 4620 cgatttccta
tttgtctctg cttattttct gattctgctc agctatgtca cttcctgcct 4680
ggccaatcag ccaatcagtg ttttattcat tagccaataa aagaaacatt tacacagaag
4740 gacttccccc atcatgttat ttgtatgagt tcttcagaaa atcatagtat
cttttaatac 4800 taatttttat aaaaaattaa ttgtattgaa aattatgtgt
atatgtgtct gtgtgtcgat 4860 ttgtgctcat aagtagcatg gagtgcagaa
gagggaatca gatctttttt taagggacaa 4920 agagtttatt cagattacat
tttaaggtga taatgtatga ttgcaaggtt atcaacatgg 4980 cagaaatgtg
aagaagctgg tcacattaca tccagagtca agagtagaga gcaatgaatt 5040
gatgcatgca ttcctgtgct cagctcactt ttcctggagc tgagctgatt gtaagccatc
5100 tgatgtcttt gctgggaact aactcaaagg caagttcaaa acctgttctt
aagtataagc 5160 catctctcca gtccctcata tggtctctta agacactttc
tttatattct tgtacataga 5220 aattgaattc ctaacaactg cattcaaatt
acaaaatagt ttttaaaagc tgatataata 5280 aatgtaaata caatctagaa
catttttata aataagcata ttaactcagt aaaaataaat 5340 gcatggttat
tttccttcat tagggaagta tgtctcccca ggctgttctc tagattctac 5400
tagtaatgct gtttgtacac catccacagg ggttttattt taaagctaag acatgaatga
5460 tggacatgct tgttagcatt tagacttttt tccttactat aattgagcta
gtatttttgt 5520 gctcagtttg atatctgtta attcagataa atgtaatagt
aggtaatttc tttgtgataa 5580 aggcatataa attgaagttg gaaaacaaaa
gcctgaaatg acagttttta agattcagaa 5640 caataatttt caaaagcagt
tacccaactt tccaaataca atctgcagtt ttcttgatat 5700 gtgataaatt
tagacaaaga aatagcacat tttaaaatag ctatttactc ttgatttttt 5760
tttcaaattt aggctagttc actagttgtg tgtaaggtta tggctgcaaa catctttgac
5820 tcttggttag ggaatccagg atgatttacg tgtttggcca aaatcttgtt
ccattctggg 5880 tttcttctct atctaggtag ctagcacaag ttaaaggtgt
ggtagtattg gaaggctctc 5940 aggtatatat ttctatattc tgtatttttt
tcctctgtca tatatttgct ttctgtttta 6000 ttgatttcta ctgttagttt
gatacttact ttcttacact ttctttggga tttattttgc 6060 tgttctaaga
tttcttagca agttcatatc actgatttta acagttgctt cttttgtaat 6120
atagactgaa tgccccttat ttgaaatgct tgggatcaga aactcagatt tgaacttttc
6180 ttttttaata tttccatcaa gtttaccagc tgaatgtcct gatccaagaa
tatgaaatct 6240 gaaatgcttt gaaatctgaa acttttagag tgataaagct
tccctttaaa ttaatttgtg 6300 ttctatattt tttgacaatg tcaacctttc
attgttatcc aatgagtgaa catattttca 6360 atttttttgt ttgatctgtt
atattttgat ctgaccatat ttataaaatt ttatttaatt 6420 tgaatgttgt
gctgttactt atctttatta ttatttttgc ttattttcta gccaaatgaa 6480
attatattct gtattatttt agtttgaatt ttactttgtg gcttagtaac tgccttttgt
6540 tggtgaatgc ttaagaaaaa cgtgtggtct actgatattg gttctaatct
tatatagcat 6600 gttgtttgtt aggtagttga ttatgctggt cagattgtct
tgagtttatg caaatgtaaa 6660 atatttagat gcttgttttg ttgtctaaga
acaaagtatg cttgctgtct cctatcggtt 6720 ctggtttttc cattcatctc
ttcaagctgt tttgtgtgtt gaatactaac tccgtactat 6780 cttgttttct
gtgaattaac cccttttcaa aggtttcttt tctttttttt tttaagggac 6840
aacaagttta ttcagattac attttaagct gataatgtat gattgcaagg ttatcaacat
6900 ggcagaaatg tgaagaagct aggcacatta catccacatg gagtcaagag
cagagagcag 6960 tgaattaatg catgcattcc tgtggtcagc tcacttttcc
tattcttaga tagtctagga 7020 tcataaacct ggggaatagt gctaccacaa
tgggcatatc cacttacttc agttcatgca 7080 atcaaccaag gcacatccac
aggaaaaact gatttagaca acctctcatt gagactcttc 7140 ccagatgatt
agactgtgtc aagttgacaa ttaaaactat cacacctgaa gccatcacta 7200
gtaaatataa tgaaaatgtt gattatcacc ataattcatc tgtatccctt tgttattgta
7260 gattttgtga agttcctatt caagtccctg ttccttcctt aaaaacctgt
tttttagtta 7320 aataggtttt ttagtgttcc tgtctgtaaa tactttttta
aagttagata ttattttcaa 7380 gtatgttctc ccagtctttg gcttgtattt
tcatcccttc aatacatata tttttgtaat 7440 ttattttttt tatttaaatt
agaaacaaag ctgcttttac atgtcagtct cagttccctc 7500 tccctcccct
cctcccctgc tccccaccta agccccaatt ccaactcctt tcttctcccc 7560
aggaagggtg aggccctcca tgggggaaat cttcaatgtc tgtcatatca tttggagcag
7620 ggcctagacc ctccccagtg tgtctaggct gagagagtat ccctctatgt
ggagagggct 7680 cccaaagttc atttgtgtac taggggtaaa tactgatcca
ctatcagtgg ccccatagat 7740 tgtccggacc tccaaactga cttcctcctt
cagggagtct ggaacagttc tatgctggtt 7800 tcccagatat cagtctgggg
tccatgagca accccttgtt caggtcagtt gtttctgtag 7860 gtttccccag
cccggtcttg acccctttgc tcatcacttc tccctctctg caactggatt 7920
ccagagttca gctcagtgtt tagctgtggg tgtctgcatc tgcttccatc agctactgga
7980 tgagggctct aggatggcat ataaggtagt catcagtctc attatcagag
aagggctttt 8040 aaggtagcct cttgattatt gcttagattg ttagttgggg
tcaaccttgt aggtctctgg 8100 acagtgacag aattctcttt aaacctataa
tggctccctc tgtggtggta tcccttttct 8160 tgctctcatc cgttcctccc
ctgactagat cttcctgctc cctcatgtcc tcctctcccc 8220 tccccttctc
cccttctctt tcttctaact ccctctcccc tccacccacg atccccatta 8280
gcttatgaga tcttgtcctt attttagcaa aacctttttg gctataaaat taattaattt
8340 aatatgctta tatcaggttt attttggcta gtatttgtat gtgtttggtt
agtgttttta 8400 accttaattg acatgtatcc ttatatttag acacagattt
aaatatttga agtttttttt 8460 tttttttttt ttaaagattt atttattttt
tatgtcttct gcctgcatgc cagaagaggg 8520 caccagatct cattcaaggt
ggttgtgagc caccatgtgg ttgctgggaa ttgaactcag 8580 gacctctgga
agaacagtca gtgctcttaa ccgctgagcc atctctccag cccctgaagt 8640
gtttctttta aagaggatag cagtgcatca tttttccctt tgaccaatga ctcctacctt
8700 actgaattgt tttagccatt tatatgtaat gctgttacca ggtttacatt
ttcttttatc 8760 ttgctaaatt tcttccctgt ttgtctcatc tcttattttt
gtctgttgga ttatataggc 8820 ttttattttt ctgtttttac agtaagttat
atcaaattaa aattatttta tggaatgggt 8880 gtgttgacta catgtatgtc
tgtgcaccat gtgctgacct ggtcttggcc agaagaaggt 8940 gtcatattct
ctgaaactgg tattgtggat gttacgaact gccatagggt gctaggaatc 9000
aaaccccagc tcctctggaa aagcagccac tgctctgagc cactgagtcc tctcttcaag
9060 caggtgatgc caacttttaa tggttaccag tggataagag tgcttgtatc
tctagcaccc 9120 atgaaaattt atgcattgct atatgggctt gtcacttcag
cattgtgtga cagagacagg 9180 aggatcccaa gagctc 9196 4 25 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
DNA 4 actcatcttg gaatctcaga attgg 25 5 24 DNA Artificial Sequence
Description of Artificial Sequence Synthetic DNA 5 cttgaccgtt
tctatcttct ctcg 24 6 979 DNA Cricetulus griseus 6 actcatcttg
gaatctcaga attggcgcta tgctactgga ggatgggaga ctgtgtttag 60
acctgtaagt gagacatgca cagacaggtc tggcctctcc actggacact ggtcaggtga
120 agtgaaggac aaaaatgttc aagtggtcga gctccccatt gtagacagcc
tccatcctcg 180 tcctccttac ttacccttgg ctgtaccaga agaccttgca
gatcgactcc tgagagtcca 240 tggtgatcct gcagtgtggt gggtatccca
gtttgtcaaa tacttgatcc gtccacaacc 300 ttggctggaa agggaaatag
aagaaaccac caagaagctt ggcttcaaac atccagttat 360 tggagtccat
gtcagacgca ctgacaaagt gggaacagaa gcagccttcc atcccattga 420
ggaatacatg gtacacgttg aagaacattt tcagcttctc gaacgcagaa tgaaagtgga
480 taaaaaaaga gtgtatctgg ccactgatga cccttctttg ttaaaggagg
caaagacaaa 540 gtactccaat tatgaattta ttagtgataa ctctatttct
tggtcagctg gactacacaa 600 ccgatacaca gaaaattcac ttcggggcgt
gatcctggat atacactttc tctcccaggc 660 tgacttcctt gtgtgtactt
tttcatccca ggtctgtagg gttgcttatg aaatcatgca 720 aacactgcat
cctgatgcct ctgcaaactt ccattcttta gatgacatct actattttgg 780
aggccaaaat gcccacaacc agattgcagt ttatcctcac caacctcgaa ctaaagagga
840 aatccccatg gaacctggag atatcattgg tgtggctgga aaccattgga
atggttactc 900 taaaggtgtc aacagaaaac taggaaaaac aggcctgtac
ccttcctaca aagtccgaga 960 gaagatagaa acggtcaag 979 7 979 DNA Rattus
norvegicus 7 actcatcttg gaatctcaga attggcgcta tgctactggt ggatgggaga
ctgtgtttag 60 acctgtaagt gagacatgca cagacagatc tggcctctcc
actggacact ggtcaggtga 120 agtgaatgac aaaaatattc aagtggtgga
gctccccatt gtagacagcc ttcatcctcg 180 gcctccttac ttaccactgg
ctgttccaga agaccttgca gatcgactcg taagagtcca 240 tggtgatcct
gcagtgtggt gggtgtccca gttcgtcaaa tatttgattc gtccacaacc 300
ttggctagaa aaggaaatag aagaagccac caagaagctt ggcttcaaac atccagtcat
360 tggagtccat gtcagacgca cagacaaagt gggaacagag gcagccttcc
atcccatcga 420 agagtacatg gtacatgttg aagaacattt tcagcttctc
gcacgcagaa tgcaagtgga 480 taaaaaaaga gtatatctgg ctaccgatga
ccctgctttg ttaaaggagg caaagacaaa 540 gtactccaat tatgaattta
ttagtgataa ctctatttct tggtcagctg gactacacaa 600
tcggtacaca gaaaattcac ttcggggcgt gatcctggat atacactttc tctctcaggc
660 tgacttccta gtgtgtactt tttcatccca ggtctgtcgg gttgcttatg
aaatcatgca 720 aaccctgcat cctgatgcct ctgcaaactt ccactcttta
gatgacatct actattttgg 780 aggccaaaat gcccacaacc agattgccgt
ttatcctcac aaacctcgaa ctgatgagga 840 aattccaatg gaacctggag
atatcattgg tgtggctgga aaccattggg atggttattc 900 taaaggtgtc
aacagaaaac ttggaaaaac aggcttatat ccctcctaca aagtccgaga 960
gaagatagaa acggtcaag 979 8 40 DNA Artificial Sequence Description
of Artificial Sequence Synthetic DNA 8 aagtataagc ttacatggat
gacgatatcg ctgcgctcgt 40 9 40 DNA Artificial Sequence Description
of Artificial Sequence Synthetic DNA 9 atttaactgc aggaagcatt
tgcggtggac gatggagggg 40 10 40 DNA Artificial Sequence Description
of Artificial Sequence Synthetic DNA 10 atttaaggta ccgaagcatt
tgcggtgcac gatggagggg 40 11 23 DNA Artificial Sequence Description
of Artificial Sequence Synthetic DNA 11 ctccaattat gaatttatta gtg
23 12 25 DNA Artificial Sequence Description of Artificial Sequence
Synthetic DNA 12 ggatgtttga agccaagctt cttgg 25 13 24 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
DNA 13 gtccatggtg atcctgcagt gtgg 24 14 23 DNA Artificial Sequence
Description of Artificial Sequence Synthetic DNA 14 caccaatgat
atctccaggt tcc 23 15 24 DNA Artificial Sequence Description of
Artificial Sequence Synthetic DNA 15 gatatcgctg cgctcgttgt cgac 24
16 24 DNA Artificial Sequence Description of Artificial Sequence
Synthetic DNA 16 caggaaggaa ggctggaaaa gagc 24 17 24 DNA Artificial
Sequence Description of Artificial Sequence Synthetic DNA 17
gatatcgctg cgctcgtcgt cgac 24 18 24 DNA Artificial Sequence
Description of Artificial Sequence Synthetic DNA 18 caggaaggaa
ggctggaaga gagc 24 19 321 PRT Cricetulus griseus 19 Met Gly Glu Pro
Gln Gly Ser Arg Arg Ile Leu Val Thr Gly Gly Ser 1 5 10 15 Gly Leu
Val Gly Arg Ala Ile Gln Lys Val Val Ala Asp Gly Ala Gly 20 25 30
Leu Pro Gly Glu Glu Trp Val Phe Val Ser Ser Lys Asp Ala Asp Leu 35
40 45 Thr Asp Ala Ala Gln Thr Gln Ala Leu Phe Gln Lys Val Gln Pro
Thr 50 55 60 His Val Ile His Leu Ala Ala Met Val Gly Gly Leu Phe
Arg Asn Ile 65 70 75 80 Lys Tyr Asn Leu Asp Phe Trp Arg Lys Asn Val
His Ile Asn Asp Asn 85 90 95 Val Leu His Ser Ala Phe Glu Val Gly
Thr Arg Lys Val Val Ser Cys 100 105 110 Leu Ser Thr Cys Ile Phe Pro
Asp Lys Thr Thr Tyr Pro Ile Asp Glu 115 120 125 Thr Met Ile His Asn
Gly Pro Pro His Ser Ser Asn Phe Gly Tyr Ser 130 135 140 Tyr Ala Lys
Arg Met Ile Asp Val Gln Asn Arg Ala Tyr Phe Gln Gln 145 150 155 160
His Gly Cys Thr Phe Thr Ala Val Ile Pro Thr Asn Val Phe Gly Pro 165
170 175 His Asp Asn Phe Asn Ile Glu Asp Gly His Val Leu Pro Gly Leu
Ile 180 185 190 His Lys Val His Leu Ala Lys Ser Asn Gly Ser Ala Leu
Thr Val Trp 195 200 205 Gly Thr Gly Lys Pro Arg Arg Gln Phe Ile Tyr
Ser Leu Asp Leu Ala 210 215 220 Arg Leu Phe Ile Trp Val Leu Arg Glu
Tyr Asn Glu Val Glu Pro Ile 225 230 235 240 Ile Leu Ser Val Gly Glu
Glu Asp Glu Val Ser Ile Lys Glu Ala Ala 245 250 255 Glu Ala Val Val
Glu Ala Met Asp Phe Cys Gly Glu Val Thr Phe Asp 260 265 270 Ser Thr
Lys Ser Asp Gly Gln Tyr Lys Lys Thr Ala Ser Asn Gly Lys 275 280 285
Leu Arg Ala Tyr Leu Pro Asp Phe Arg Phe Thr Pro Phe Lys Gln Ala 290
295 300 Val Lys Glu Thr Cys Ala Trp Phe Thr Asp Asn Tyr Glu Gln Ala
Arg 305 310 315 320 Lys 20 590 PRT Cricetulus griseus 20 Met Ala
Ser Leu Arg Glu Ala Ser Leu Arg Lys Leu Arg Arg Phe Ser 1 5 10 15
Glu Met Arg Gly Lys Pro Val Ala Thr Gly Lys Phe Trp Asp Val Val 20
25 30 Val Ile Thr Ala Ala Asp Glu Lys Gln Glu Leu Ala Tyr Lys Gln
Gln 35 40 45 Leu Ser Glu Lys Leu Lys Arg Lys Glu Leu Pro Leu Gly
Val Asn Tyr 50 55 60 His Val Phe Thr Asp Pro Pro Gly Thr Lys Ile
Gly Asn Gly Gly Ser 65 70 75 80 Thr Leu Cys Ser Leu Gln Cys Leu Glu
Ser Leu Tyr Gly Asp Lys Trp 85 90 95 Asn Ser Phe Thr Val Leu Leu
Ile His Ser Gly Gly Tyr Ser Gln Arg 100 105 110 Leu Pro Asn Ala Ser
Ala Leu Gly Lys Ile Phe Thr Ala Leu Pro Leu 115 120 125 Gly Glu Pro
Ile Tyr Gln Met Leu Asp Leu Lys Leu Ala Met Tyr Met 130 135 140 Asp
Phe Pro Ser Arg Met Lys Pro Gly Val Leu Val Thr Cys Ala Asp 145 150
155 160 Asp Ile Glu Leu Tyr Ser Ile Gly Asp Ser Glu Ser Ile Ala Phe
Glu 165 170 175 Gln Pro Gly Phe Thr Ala Leu Ala His Pro Ser Ser Leu
Ala Val Gly 180 185 190 Thr Thr His Gly Val Phe Val Leu Asp Ser Ala
Gly Ser Leu Gln His 195 200 205 Gly Asp Leu Glu Tyr Arg Gln Cys His
Arg Phe Leu His Lys Pro Ser 210 215 220 Ile Glu Asn Met His His Phe
Asn Ala Val His Arg Leu Gly Ser Phe 225 230 235 240 Gly Gln Gln Asp
Leu Ser Gly Gly Asp Thr Thr Cys His Pro Leu His 245 250 255 Ser Glu
Tyr Val Tyr Thr Asp Ser Leu Phe Tyr Met Asp His Lys Ser 260 265 270
Ala Lys Lys Leu Leu Asp Phe Tyr Glu Ser Val Gly Pro Leu Asn Cys 275
280 285 Glu Ile Asp Ala Tyr Gly Asp Phe Leu Gln Ala Leu Gly Pro Gly
Ala 290 295 300 Thr Ala Glu Tyr Thr Lys Asn Thr Ser His Val Thr Lys
Glu Glu Ser 305 310 315 320 His Leu Leu Asp Met Arg Gln Lys Ile Phe
His Leu Leu Lys Gly Thr 325 330 335 Pro Leu Asn Val Val Val Leu Asn
Asn Ser Arg Phe Tyr His Ile Gly 340 345 350 Thr Thr Glu Glu Tyr Leu
Leu His Phe Thr Ser Asn Gly Ser Leu Gln 355 360 365 Ala Glu Leu Gly
Leu Gln Ser Ile Ala Phe Ser Val Phe Pro Asn Val 370 375 380 Pro Glu
Asp Ser His Glu Lys Pro Cys Val Ile His Ser Ile Leu Asn 385 390 395
400 Ser Gly Cys Cys Val Ala Pro Gly Ser Val Val Glu Tyr Ser Arg Leu
405 410 415 Gly Pro Glu Val Ser Ile Ser Glu Asn Cys Ile Ile Ser Gly
Ser Val 420 425 430 Ile Glu Lys Ala Val Leu Pro Pro Cys Ser Phe Val
Cys Ser Leu Ser 435 440 445 Val Glu Ile Asn Gly His Leu Glu Tyr Ser
Thr Met Val Phe Gly Met 450 455 460 Glu Asp Asn Leu Lys Asn Ser Val
Lys Thr Ile Ser Asp Ile Lys Met 465 470 475 480 Leu Gln Phe Phe Gly
Val Cys Phe Leu Thr Cys Leu Asp Ile Trp Asn 485 490 495 Leu Lys Ala
Met Glu Glu Leu Phe Ser Gly Ser Lys Thr Gln Leu Ser 500 505 510 Leu
Trp Thr Ala Arg Ile Phe Pro Val Cys Ser Ser Leu Ser Glu Ser 515 520
525 Val Ala Ala Ser Leu Gly Met Leu Asn Ala Ile Arg Asn His Ser Pro
530 535 540 Phe Ser Leu Ser Asn Phe Lys Leu Leu Ser Ile Gln Glu Met
Leu Leu 545 550 555 560 Cys Lys Asp Val Gly Asp Met Leu Ala Tyr Arg
Glu Gln Leu Phe Leu 565 570 575 Glu Ile Ser Ser Lys Arg Lys Gln Ser
Asp Ser Glu Lys Ser 580 585 590 21 25 PRT Homo sapiens 21 Gln Val
Thr Val Gln Ser Ser Pro Asn Phe Thr Gln His Val Arg Glu 1 5 10 15
Gln Ser Leu Val Thr Asp Gln Leu Cys 20 25 22 32 DNA Artificial
Sequence Description of Artificial Sequence Synthetic DNA 22
taaatagaat tcggcatcat gtggcagctg ct 32 23 575 PRT Cricetulus
griseus 23 Met Arg Ala Trp Thr Gly Ser Trp Arg Trp Ile Met Leu Ile
Leu Phe 1 5 10 15 Ala Trp Gly Thr Leu Leu Phe Tyr Ile Gly Gly His
Leu Val Arg Asp 20 25 30 Asn Asp His Pro Asp His Ser Ser Arg Glu
Leu Ser Lys Ile Leu Ala 35 40 45 Lys Leu Glu Arg Leu Lys Gln Gln
Asn Glu Asp Leu Arg Arg Met Ala 50 55 60 Glu Ser Leu Arg Ile Pro
Glu Gly Pro Ile Asp Gln Gly Thr Ala Thr 65 70 75 80 Gly Arg Val Arg
Val Leu Glu Glu Gln Leu Val Lys Ala Lys Glu Gln 85 90 95 Ile Glu
Asn Tyr Lys Lys Gln Ala Arg Asn Asp Leu Gly Lys Asp His 100 105 110
Glu Ile Leu Arg Arg Arg Ile Glu Asn Gly Ala Lys Glu Leu Trp Phe 115
120 125 Phe Leu Gln Ser Glu Leu Lys Lys Leu Lys Lys Leu Glu Gly Asn
Glu 130 135 140 Leu Gln Arg His Ala Asp Glu Ile Leu Leu Asp Leu Gly
His His Glu 145 150 155 160 Arg Ser Ile Met Thr Asp Leu Tyr Tyr Leu
Ser Gln Thr Asp Gly Ala 165 170 175 Gly Glu Trp Arg Glu Lys Glu Ala
Lys Asp Leu Thr Glu Leu Val Gln 180 185 190 Arg Arg Ile Thr Tyr Leu
Gln Asn Pro Lys Asp Cys Ser Lys Ala Arg 195 200 205 Lys Leu Val Cys
Asn Ile Asn Lys Gly Cys Gly Tyr Gly Cys Gln Leu 210 215 220 His His
Val Val Tyr Cys Phe Met Ile Ala Tyr Gly Thr Gln Arg Thr 225 230 235
240 Leu Ile Leu Glu Ser Gln Asn Trp Arg Tyr Ala Thr Gly Gly Trp Glu
245 250 255 Thr Val Phe Arg Pro Val Ser Glu Thr Cys Thr Asp Arg Ser
Gly Leu 260 265 270 Ser Thr Gly His Trp Ser Gly Glu Val Lys Asp Lys
Asn Val Gln Val 275 280 285 Val Glu Leu Pro Ile Val Asp Ser Leu His
Pro Arg Pro Pro Tyr Leu 290 295 300 Pro Leu Ala Val Pro Glu Asp Leu
Ala Asp Arg Leu Leu Arg Val His 305 310 315 320 Gly Asp Pro Ala Val
Trp Trp Val Ser Gln Phe Val Lys Tyr Leu Ile 325 330 335 Arg Pro Gln
Pro Trp Leu Glu Arg Glu Ile Glu Glu Thr Thr Lys Lys 340 345 350 Leu
Gly Phe Lys His Pro Val Ile Gly Val His Val Arg Arg Thr Asp 355 360
365 Lys Val Gly Thr Glu Ala Ala Phe His Pro Ile Glu Glu Tyr Met Val
370 375 380 His Val Glu Glu His Phe Gln Leu Leu Glu Arg Arg Met Lys
Val Asp 385 390 395 400 Lys Lys Arg Val Tyr Leu Ala Thr Asp Asp Pro
Ser Leu Leu Lys Glu 405 410 415 Ala Lys Thr Lys Tyr Ser Asn Tyr Glu
Phe Ile Ser Asp Asn Ser Ile 420 425 430 Ser Trp Ser Ala Gly Leu His
Asn Arg Tyr Thr Glu Asn Ser Leu Arg 435 440 445 Gly Val Ile Leu Asp
Ile His Phe Leu Ser Gln Ala Asp Phe Leu Val 450 455 460 Cys Thr Phe
Ser Ser Gln Val Cys Arg Val Ala Tyr Glu Ile Met Gln 465 470 475 480
Thr Leu His Pro Asp Ala Ser Ala Asn Phe His Ser Leu Asp Asp Ile 485
490 495 Tyr Tyr Phe Gly Gly Gln Asn Ala His Asn Gln Ile Ala Val Tyr
Pro 500 505 510 His Gln Pro Arg Thr Lys Glu Glu Ile Pro Met Glu Pro
Gly Asp Ile 515 520 525 Ile Gly Val Ala Gly Asn His Trp Asn Gly Tyr
Ser Lys Gly Val Asn 530 535 540 Arg Lys Leu Gly Lys Thr Gly Leu Tyr
Pro Ser Tyr Lys Val Arg Glu 545 550 555 560 Lys Ile Glu Thr Val Lys
Tyr Pro Thr Tyr Pro Glu Ala Glu Lys 565 570 575 24 575 PRT Mus
musculus 24 Met Arg Ala Trp Thr Gly Ser Trp Arg Trp Ile Met Leu Ile
Leu Phe 1 5 10 15 Ala Trp Gly Thr Leu Leu Phe Tyr Ile Gly Gly His
Leu Val Arg Asp 20 25 30 Asn Asp His Pro Asp His Ser Ser Arg Glu
Leu Ser Lys Ile Leu Ala 35 40 45 Lys Leu Glu Arg Leu Lys Gln Gln
Asn Glu Asp Leu Arg Arg Met Ala 50 55 60 Glu Ser Leu Arg Ile Pro
Glu Gly Pro Ile Asp Gln Gly Thr Ala Thr 65 70 75 80 Gly Arg Val Arg
Val Leu Glu Glu Gln Leu Val Lys Ala Lys Glu Gln 85 90 95 Ile Glu
Asn Tyr Lys Lys Gln Ala Arg Asn Gly Leu Gly Lys Asp His 100 105 110
Glu Ile Leu Arg Arg Arg Ile Glu Asn Gly Ala Lys Glu Leu Trp Phe 115
120 125 Phe Leu Gln Ser Glu Leu Lys Lys Leu Lys His Leu Glu Gly Asn
Glu 130 135 140 Leu Gln Arg His Ala Asp Glu Ile Leu Leu Asp Leu Gly
His His Glu 145 150 155 160 Arg Ser Ile Met Thr Asp Leu Tyr Tyr Leu
Ser Gln Thr Asp Gly Ala 165 170 175 Gly Asp Trp Arg Glu Lys Glu Ala
Lys Asp Leu Thr Glu Leu Val Gln 180 185 190 Arg Arg Ile Thr Tyr Leu
Gln Asn Pro Lys Asp Cys Ser Lys Ala Arg 195 200 205 Lys Leu Val Cys
Asn Ile Asn Lys Gly Cys Gly Tyr Gly Cys Gln Leu 210 215 220 His His
Val Val Tyr Cys Phe Met Ile Ala Tyr Gly Thr Gln Arg Thr 225 230 235
240 Leu Ile Leu Glu Ser Gln Asn Trp Arg Tyr Ala Thr Gly Gly Trp Glu
245 250 255 Thr Val Phe Arg Pro Val Ser Glu Thr Cys Thr Asp Arg Ser
Gly Leu 260 265 270 Ser Thr Gly His Trp Ser Gly Glu Val Asn Asp Lys
Asn Ile Gln Val 275 280 285 Val Glu Leu Pro Ile Val Asp Ser Leu His
Pro Arg Pro Pro Tyr Leu 290 295 300 Pro Leu Ala Val Pro Glu Asp Leu
Ala Asp Arg Leu Leu Arg Val His 305 310 315 320 Gly Asp Pro Ala Val
Trp Trp Val Ser Gln Phe Val Lys Tyr Leu Ile 325 330 335 Arg Pro Gln
Pro Trp Leu Glu Lys Glu Ile Glu Glu Ala Thr Lys Lys 340 345 350 Leu
Gly Phe Lys His Pro Val Ile Gly Val His Val Arg Arg Thr Asp 355 360
365 Lys Val Gly Thr Glu Ala Ala Phe His Pro Ile Glu Glu Tyr Met Val
370 375 380 His Val Glu Glu His Phe Gln Leu Leu Ala Arg Arg Met Gln
Val Asp 385 390 395 400 Lys Lys Arg Val Tyr Leu Ala Thr Asp Asp Pro
Thr Leu Leu Lys Glu 405 410 415 Ala Lys Thr Lys Tyr Ser Asn Tyr Glu
Phe Ile Ser Asp Asn Ser Ile 420 425 430 Ser Trp Ser Ala Gly Leu His
Asn Arg Tyr Thr Glu Asn Ser Leu Arg 435 440 445 Gly Val Ile Leu Asp
Ile His Phe Leu Ser Gln Ala Asp Phe Leu Val 450 455 460 Cys Thr Phe
Ser Ser Gln Val Cys Arg Val Ala Tyr Glu Ile Met Gln 465 470 475 480
Thr Leu His Pro Asp Ala Ser Ala Asn Phe His Ser Leu Asp Asp Ile 485
490 495 Tyr Tyr Phe Gly Gly Gln Asn Ala His Asn Gln Ile Ala Val Tyr
Pro 500 505 510 His Lys Pro Arg Thr Glu Glu Glu Ile Pro Met Glu Pro
Gly Asp Ile 515 520 525 Ile Gly Val Ala Gly Asn His Trp Asp Gly Tyr
Ser Lys Gly Ile Asn 530 535 540 Arg Lys Leu Gly Lys Thr Gly Leu Tyr
Pro Ser Tyr Lys Val Arg Glu 545 550 555 560 Lys Ile Glu Thr Val Lys
Tyr Pro Thr Tyr Pro Glu Ala Glu Lys 565 570 575 25 18 PRT Homo
sapiens 25 Asp Glu
Ser Ile Tyr Ser Asn Tyr Tyr Leu Tyr Glu Ser Ile Pro Lys 1 5 10 15
Pro Cys 26 34 DNA Artificial Sequence Description of Artificial
Sequence Synthetic DNA 26 aataaaggat cctggggtca tttgtcttga gggt 34
27 788 DNA Homo sapiens 27 gaa ttc ggc atc atg tgg cag ctg ctc ctc
cca act gct ctg cta ctt 48 Met Trp Gln Leu Leu Leu Pro Thr Ala Leu
Leu Leu 1 5 10 cta gtt tca gct ggc atg cgg act gaa gat ctc cca aag
gct gtg gtg 96 Leu Val Ser Ala Gly Met Arg Thr Glu Asp Leu Pro Lys
Ala Val Val 15 20 25 ttc ctg gag cct caa tgg tac agg gtg ctc gag
aag gac agt gtg act 144 Phe Leu Glu Pro Gln Trp Tyr Arg Val Leu Glu
Lys Asp Ser Val Thr 30 35 40 ctg aag tgc cag gga gcc tac tcc cct
gag gac aat tcc aca cag tgg 192 Leu Lys Cys Gln Gly Ala Tyr Ser Pro
Glu Asp Asn Ser Thr Gln Trp 45 50 55 60 ttt cac aat gag agc ctc atc
tca agc cag gcc tcg agc tac ttc att 240 Phe His Asn Glu Ser Leu Ile
Ser Ser Gln Ala Ser Ser Tyr Phe Ile 65 70 75 gac gct gcc aca gtc
gac gac agt gga gag tac agg tgc cag aca aac 288 Asp Ala Ala Thr Val
Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn 80 85 90 ctc tcc acc
ctc agt gac ccg gtg cag cta gaa gtc cat atc ggc tgg 336 Leu Ser Thr
Leu Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp 95 100 105 ctg
ttg ctc cag gcc cct cgg tgg gtg ttc aag gag gaa gac cct att 384 Leu
Leu Leu Gln Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile 110 115
120 cac ctg agg tgt cac agc tgg aag aac act gct ctg cat aag gtc aca
432 His Leu Arg Cys His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr
125 130 135 140 tat tta cag aat ggc aaa ggc agg aag tat ttt cat cat
aat tct gac 480 Tyr Leu Gln Asn Gly Lys Gly Arg Lys Tyr Phe His His
Asn Ser Asp 145 150 155 ttc tac att cca aaa gcc aca ctc aaa gac agc
ggc tcc tac ttc tgc 528 Phe Tyr Ile Pro Lys Ala Thr Leu Lys Asp Ser
Gly Ser Tyr Phe Cys 160 165 170 agg ggg ctt ttt ggg agt aaa aat gtg
tct tca gag act gtg aac atc 576 Arg Gly Leu Phe Gly Ser Lys Asn Val
Ser Ser Glu Thr Val Asn Ile 175 180 185 acc atc act caa ggt ttg gca
gtg tca acc atc tca tca ttc ttt cca 624 Thr Ile Thr Gln Gly Leu Ala
Val Ser Thr Ile Ser Ser Phe Phe Pro 190 195 200 cct ggg tac caa gtc
tct ttc tgc ttg gtg atg gta ctc ctt ttt gca 672 Pro Gly Tyr Gln Val
Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala 205 210 215 220 gtg gac
aca gga cta tat ttc tct gtg aag aca aac att cga agc tca 720 Val Asp
Thr Gly Leu Tyr Phe Ser Val Lys Thr Asn Ile Arg Ser Ser 225 230 235
aca aga gac tgg aag gac cat aaa ttt aaa tgg aga aag gac cct caa 768
Thr Arg Asp Trp Lys Asp His Lys Phe Lys Trp Arg Lys Asp Pro Gln 240
245 250 gac aaa tga ccc cag gat cc 788 Asp Lys 28 254 PRT Homo
sapiens 28 Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val
Ser Ala 1 5 10 15 Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val
Phe Leu Glu Pro 20 25 30 Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser
Val Thr Leu Lys Cys Gln 35 40 45 Gly Ala Tyr Ser Pro Glu Asp Asn
Ser Thr Gln Trp Phe His Asn Glu 50 55 60 Ser Leu Ile Ser Ser Gln
Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr 65 70 75 80 Val Asp Asp Ser
Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu 85 90 95 Ser Asp
Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 100 105 110
Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 115
120 125 His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln
Asn 130 135 140 Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe
Tyr Ile Pro 145 150 155 160 Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr
Phe Cys Arg Gly Leu Phe 165 170 175 Gly Ser Lys Asn Val Ser Ser Glu
Thr Val Asn Ile Thr Ile Thr Gln 180 185 190 Gly Leu Ala Val Ser Thr
Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln 195 200 205 Val Ser Phe Cys
Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly 210 215 220 Leu Tyr
Phe Ser Val Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp 225 230 235
240 Lys Asp His Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys 245 250
29 51 DNA Artificial Sequence Description of Artificial Sequence
Synthetic DNA 29 tgttggatcc tgtcaatgat gatgatgatg atgaccttga
gtgatggtga t 51 30 620 DNA Homo sapiens 30 gaa ttc ggc atc atg tgg
cag ctg ctc ctc cca act gct ctg cta ctt 48 Met Trp Gln Leu Leu Leu
Pro Thr Ala Leu Leu Leu 1 5 10 cta gtt tca gct ggc atg cgg act gaa
gat ctc cca aag gct gtg gtg 96 Leu Val Ser Ala Gly Met Arg Thr Glu
Asp Leu Pro Lys Ala Val Val 15 20 25 ttc ctg gag cct caa tgg tac
agg gtg ctc gag aag gac agt gtg act 144 Phe Leu Glu Pro Gln Trp Tyr
Arg Val Leu Glu Lys Asp Ser Val Thr 30 35 40 ctg aag tgc cag gga
gcc tac tcc cct gag gac aat tcc aca cag tgg 192 Leu Lys Cys Gln Gly
Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp 45 50 55 60 ttt cac aat
gag agc ctc atc tca agc cag gcc tcg agc tac ttc att 240 Phe His Asn
Glu Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile 65 70 75 gac
gct gcc aca gtc gac gac agt gga gag tac agg tgc cag aca aac 288 Asp
Ala Ala Thr Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn 80 85
90 ctc tcc acc ctc agt gac ccg gtg cag cta gaa gtc cat atc ggc tgg
336 Leu Ser Thr Leu Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp
95 100 105 ctg ttg ctc cag gcc cct cgg tgg gtg ttc aag gag gaa gac
cct att 384 Leu Leu Leu Gln Ala Pro Arg Trp Val Phe Lys Glu Glu Asp
Pro Ile 110 115 120 cac ctg agg tgt cac agc tgg aag aac act gct ctg
cat aag gtc aca 432 His Leu Arg Cys His Ser Trp Lys Asn Thr Ala Leu
His Lys Val Thr 125 130 135 140 tat tta cag aat ggc aaa ggc agg aag
tat ttt cat cat aat tct gac 480 Tyr Leu Gln Asn Gly Lys Gly Arg Lys
Tyr Phe His His Asn Ser Asp 145 150 155 ttc tac att cca aaa gcc aca
ctc aaa gac agc ggc tcc tac ttc tgc 528 Phe Tyr Ile Pro Lys Ala Thr
Leu Lys Asp Ser Gly Ser Tyr Phe Cys 160 165 170 agg ggg ctt ttt ggg
agt aaa aat gtg tct tca gag act gtg aac atc 576 Arg Gly Leu Phe Gly
Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile 175 180 185 acc atc act
caa ggt cat cat cat cat cat cat tga cag gat cc 620 Thr Ile Thr Gln
Gly His His His His His His 190 195 31 199 PRT Homo sapiens 31 Met
Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala 1 5 10
15 Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro
20 25 30 Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys
Cys Gln 35 40 45 Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp
Phe His Asn Glu 50 55 60 Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr
Phe Ile Asp Ala Ala Thr 65 70 75 80 Val Asp Asp Ser Gly Glu Tyr Arg
Cys Gln Thr Asn Leu Ser Thr Leu 85 90 95 Ser Asp Pro Val Gln Leu
Glu Val His Ile Gly Trp Leu Leu Leu Gln 100 105 110 Ala Pro Arg Trp
Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 115 120 125 His Ser
Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn 130 135 140
Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro 145
150 155 160 Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly
Leu Phe 165 170 175 Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile
Thr Ile Thr Gln 180 185 190 Gly His His His His His His 195 32 24
DNA Artificial Sequence Description of Artificial Sequence
Synthetic DNA 32 aggaaggtgg cgctcatcac gggc 24 33 26 DNA Artificial
Sequence Description of Artificial Sequence Synthetic DNA 33
taaggccaca agtcttaatt gcatcc 26 34 27 DNA Artificial Sequence
Description of Artificial Sequence Synthetic DNA 34 caggggtgtt
cccttgagga ggtggaa 27 35 23 DNA Artificial Sequence Description of
Artificial Sequence Synthetic DNA 35 cccctcacgc atgaagcctg gag 23
36 28 DNA Artificial Sequence Description of Artificial Sequence
Synthetic DNA 36 ggcaggagac caccttgcga gtgcccac 28 37 28 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
DNA 37 ggcgctggct tacccggaga ggaatggg 28 38 28 DNA Artificial
Sequence Description of Artificial Sequence Synthetic DNA 38
aaaaggcctc agttagtgaa ctgtatgg 28 39 29 DNA Artificial Sequence
Description of Artificial Sequence Synthetic DNA 39 cgcggatcct
caagcgttgg ggttggtcc 29 40 45 DNA Artificial Sequence Description
of Artificial Sequence Synthetic DNA 40 cccaagcttg ccaccatggc
tcacgctccc gctagctgcc cgagc 45 41 31 DNA Artificial Sequence
Description of Artificial Sequence Synthetic DNA 41 ccggaattct
gccaagtatg agccatcctg g 31 42 17 DNA Artificial Sequence
Description of Artificial Sequence Synthetic DNA 42 gccatccaga
aggtggt 17 43 17 DNA Artificial Sequence Description of Artificial
Sequence Synthetic DNA 43 gtcttgtcag ggaagat 17 44 28 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
DNA 44 ggcaggagac caccttgcga gtgcccac 28 45 28 DNA Artificial
Sequence Description of Artificial Sequence Synthetic DNA 45
gggtgggctg taccttctgg aacagggc 28 46 28 DNA Artificial Sequence
Description of Artificial Sequence Synthetic DNA 46 ggcgctggct
tacccggaga ggaatggg 28 47 30 DNA Artificial Sequence Description of
Artificial Sequence Synthetic DNA 47 ggaatgggtg tttgtctcct
ccaaagatgc 30 48 1316 DNA Cricetulus griseus 48 gccccgcccc
ctccacctgg accgagagta gctggagaat tgtgcaccgg aagtagctct 60
tggactggtg gaaccctgcg caggtgcagc aacaatgggt gagccccagg gatccaggag
120 gatcctagtg acagggggct ctggactggt gggcagagct atccagaagg
tggtcgdaga 180 tggcgctggc ttacccggag aggaatgggt gtttgtctcc
tccaaagatg cagatctgac 240 ggatgcagca caaacccaag ccctgttcca
gaaggtacag cccacccatg tcatccatct 300 tgctgcaatg gtaggaggcc
ttttccggaa tatcaaatac aacttggatt tctggaggaa 360 gaatgtgcac
atcaatgaca acgtcctgca ctcagctttc gaggtgggca ctcgcaaggt 420
ggtctcctgc ctgtccacct gtatcttccc tgacaagacc acctatccta ttgatgaaac
480 aatgatccac aatggtccac cccacagcag caattttggg tactcgtatg
ccaagaggat 540 gattgacgtg cagaacaggg cctacttcca gcagcatggc
tgcaccttca ctgctgtcat 600 ccctaccaat gtctttggac ctcatgacaa
cttcaacatt gaagatggcc atgtgctgcc 660 tggcctcatc cataaggtgc
atctggccaa gagtaatggt tcagccttga ctgtttgggg 720 tacagggaaa
ccacggaggc agttcatcta ctcactggac ctagcccggc tcttcatctg 780
ggtcctgcgg gagtacaatg aagttgagcc catcatcctc tcagtgggcg aggaagatga
840 agtctccatt aaggaggcag ctgaggctgt agtggaggcc atggacttct
gtggggaagt 900 cacttttgat tcaacaaagt cagatgggca gtataagaag
acagccagca atggcaagct 960 tcgggcctac ttgcctgatt tccgtttcac
acccttcaag caggctgtga aggagacctg 1020 tgcctggttc accgacaact
atgagcaggc ccggaagtga agcatgggac aagcgggtgc 1080 tcagctggca
atgcccagtc agtaggctgc agtctcatca tttgcttgtc aagaactgag 1140
gacagtatcc agcaacctga gccacatgct ggtctctctg ccagggggct tcatgcagcc
1200 atccagtagg gcccatgttt gtccatcctc gggggaaggc cagaccaaca
ccttgtttgt 1260 ctgcttctgc cccaacctca gtgcatccat gctggtcctg
ctgtcccttg tctaga 1316 49 23 DNA Artificial Sequence Description of
Artificial Sequence Synthetic DNA 49 gatcctgctg ggaccaaaat tgg 23
50 22 DNA Artificial Sequence Description of Artificial Sequence
Synthetic DNA 50 cttaacatcc caagggatgc tg 22 51 1965 DNA Cricetulus
griseus 51 acggggggct cccggaagcg gggaccatgg cgtctctgcg cgaagcgagc
ctgcggaagc 60 tgcggcgctt ttccgagatg agaggcaaac ctgtggcaac
tgggaaattc tgggatgtag 120 ttgtaataac agcagctgac gaaaagcagg
agcttgctta caagcaacag ttgtcggaga 180 agctgaagag aaaggaattg
ccccttggag ttaactacca tgttttcact gatcctcctg 240 gaaccaaaat
tggaaatgga ggatcaacac tttgttctct tcagtgcctg gaaagcctct 300
atggagacaa gtggaattcc ttcacagtcc tgttaattca ctctggtggc tacagtcaac
360 gacttcccaa tgcaagcgct ttaggaaaaa tcttcacggc tttaccactt
ggtgagccca 420 tttatcagat gttggactta aaactagcca tgtacatgga
tttcccctca cgcatgaagc 480 ctggagtttt ggtcacctgt gcagatgata
ttgaactata cagcattggg gactctgagt 540 ccattgcatt tgagcagcct
ggctttactg ccctagccca tccatctagt ctggctgtag 600 gcaccacaca
tggagtattt gtattggact ctgccggttc tttgcaacat ggtgacctag 660
agtacaggca atgccaccgt ttcctccata agcccagcat tgaaaacatg caccacttta
720 atgccgtgca tagactagga agctttggtc aacaggactt gagtgggggt
gacaccacct 780 gtcatccatt gcactctgag tatgtctaca cagatagcct
attttacatg gatcataaat 840 cagccaaaaa gctacttgat ttctatgaaa
gtgtaggccc actgaactgt gaaatagatg 900 cctatggtga ctttctgcag
gcactgggac ctggagcaac tgcagagtac accaagaaca 960 cctcacacgt
cactaaagag gaatcacact tgttggacat gaggcagaaa atattccacc 1020
tcctcaaggg aacacccctg aatgttgttg tccttaataa ctccaggttt tatcacattg
1080 gaacaacgga ggagtatctg ctacatttca cttccaatgg ttcgttacag
gcagagctgg 1140 gcttgcaatc catagctttc agtgtctttc caaatgtgcc
tgaagactcc catgagaaac 1200 cctgtgtcat tcacagcatc ctgaattcag
gatgctgtgt ggcccctggc tcagtggtag 1260 aatattccag attaggacct
gaggtgtcca tctcggaaaa ctgcattatc agcggttctg 1320 tcatagaaaa
agctgttctg cccccatgtt ctttcgtgtg ctctttaagt gtggagataa 1380
atggacactt agaatattca actatggtgt ttggcatgga agacaacttg aagaacagtg
1440 ttaaaaccat atcagatata aagatgcttc agttctttgg agtctgtttc
ctgacttgtt 1500 tagatatttg gaaccttaaa gctatggaag aactattttc
aggaagtaag acgcagctga 1560 gcctgtggac tgctcgaatt ttccctgtct
gttcttctct gagtgagtcg gttgcagcat 1620 cccttgggat gttaaatgcc
attcgaaacc attcgccatt cagcctgagc aacttcaagc 1680 tgctgtccat
ccaggaaatg cttctctgca aagatgtagg agacatgctt gcttacaggg 1740
agcaactctt tctagaaatc agttcaaaga gaaaacagtc tgattcggag aaatcttaaa
1800 tacaatggat tttgcctgga aacaggattg caaatgcagg catattctat
agatctctgg 1860 gttcttcttt ctttctcccc tctctccttt cctttccctt
tgatgtaatg acaaaggtaa 1920 aaatggccac ttctgatgga aaaaaaaaaa
aaaaaaaaaa aaaaa 1965 52 27 DNA Artificial Sequence Description of
Artificial Sequence Synthetic DNA 52 caggggtgtt cccttgagga ggtggaa
27 53 27 DNA Artificial Sequence Description of Artificial Sequence
Synthetic DNA 53 cactgagcca ggggccacac agcatcc 27 54 23 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
DNA 54 cccctcacgc atgaagcctg gag 23 55 27 DNA Artificial Sequence
Description of Artificial Sequence Synthetic DNA 55 tgccaccgtt
tcctccataa gcccagc 27 56 28 DNA Artificial Sequence Description of
Artificial Sequence Synthetic DNA 56 atggctcaag ctcccgctaa gtgcccga
28 57 27 DNA Artificial Sequence Description of Artificial Sequence
Synthetic DNA 57 tcaagcgttt gggttggtcc tcatgag 27 58 25 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
DNA 58 tccggggatg gcgagatggg caagc 25 59 24 DNA Artificial Sequence
Description of Artificial Sequence Synthetic DNA 59 cttgacatgg
ctctgggctc caag 24 60 25 DNA Artificial Sequence Description of
Artificial Sequence Synthetic DNA 60 ccacttcagt cggtcggtag tattt 25
61 24 DNA Artificial Sequence Description of Artificial Sequence
Synthetic DNA 61 cgctcacccg cctgaggcga catg
24 62 32 DNA Artificial Sequence Description of Artificial Sequence
Synthetic DNA 62 ggcaggtgct gtcggtgagg tcaccatagt gc 32 63 24 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
DNA 63 ggggccatgc caaggactat gtcg 24 64 25 DNA Artificial Sequence
Description of Artificial Sequence Synthetic DNA 64 atgtggctga
tgttacaaaa tgatg 25 65 1504 DNA Cricetulus griseus 65 atg gct cac
gct ccc gct agc tgc ccg agc tcc agg aac tct ggg gac 48 Met Ala His
Ala Pro Ala Ser Cys Pro Ser Ser Arg Asn Ser Gly Asp 1 5 10 15 ggc
gat aag ggc aag ccc agg aag gtg gcg ctc atc acg ggc atc acc 96 Gly
Asp Lys Gly Lys Pro Arg Lys Val Ala Leu Ile Thr Gly Ile Thr 20 25
30 ggc cag gat ggc tca tac ttg gca gaa ttc ctg ctg gag aaa gga tac
144 Gly Gln Asp Gly Ser Tyr Leu Ala Glu Phe Leu Leu Glu Lys Gly Tyr
35 40 45 gag gtt cat gga att gta cgg cga tcc agt tca ttt aat aca
ggt cga 192 Glu Val His Gly Ile Val Arg Arg Ser Ser Ser Phe Asn Thr
Gly Arg 50 55 60 att gaa cat tta tat aag aat cca cag gct cat att
gaa gga aac atg 240 Ile Glu His Leu Tyr Lys Asn Pro Gln Ala His Ile
Glu Gly Asn Met 65 70 75 80 aag ttg cac tat ggt gac ctc acc gac agc
acc tgc cta gta aaa atc 288 Lys Leu His Tyr Gly Asp Leu Thr Asp Ser
Thr Cys Leu Val Lys Ile 85 90 95 atc aat gaa gtc aaa cct aca gag
atc tac aat ctt ggt gcc cag agc 336 Ile Asn Glu Val Lys Pro Thr Glu
Ile Tyr Asn Leu Gly Ala Gln Ser 100 105 110 cat gtc aag att tcc ttt
gac tta gca gag tac act gca gat gtt gat 384 His Val Lys Ile Ser Phe
Asp Leu Ala Glu Tyr Thr Ala Asp Val Asp 115 120 125 gga gtt ggc acc
ttg cgg ctt ctg gat gca att aag act tgt ggc ctt 432 Gly Val Gly Thr
Leu Arg Leu Leu Asp Ala Ile Lys Thr Cys Gly Leu 130 135 140 ata aat
tct gtg aag ttc tac cag gcc tca act agt gaa ctg tat gga 480 Ile Asn
Ser Val Lys Phe Tyr Gln Ala Ser Thr Ser Glu Leu Tyr Gly 145 150 155
160 aaa gtg caa gaa ata ccc cag aaa gag acc acc cct ttc tat cca agg
528 Lys Val Gln Glu Ile Pro Gln Lys Glu Thr Thr Pro Phe Tyr Pro Arg
165 170 175 tcg ccc tat gga gca gcc aaa ctt tat gcc tat tgg att gta
gtg aac 576 Ser Pro Tyr Gly Ala Ala Lys Leu Tyr Ala Tyr Trp Ile Val
Val Asn 180 185 190 ttt cga gag gct tat aat ctc ttt gcg gtg aac ggc
att ctc ttc aat 624 Phe Arg Glu Ala Tyr Asn Leu Phe Ala Val Asn Gly
Ile Leu Phe Asn 195 200 205 cat gag agt cct aga aga gga gct aat ttt
gtt act cga aaa att agc 672 His Glu Ser Pro Arg Arg Gly Ala Asn Phe
Val Thr Arg Lys Ile Ser 210 215 220 cgg tca gta gct aag att tac ctt
gga caa ctg gaa tgt ttc agt ttg 720 Arg Ser Val Ala Lys Ile Tyr Leu
Gly Gln Leu Glu Cys Phe Ser Leu 225 230 235 240 gga aat ctg gac gcc
aaa cga gac tgg ggc cat gcc aag gac tat gtc 768 Gly Asn Leu Asp Ala
Lys Arg Asp Trp Gly His Ala Lys Asp Tyr Val 245 250 255 gag gct atg
tgg ctg atg tta caa aat gat gaa cca gag gac ttt gtc 816 Glu Ala Met
Trp Leu Met Leu Gln Asn Asp Glu Pro Glu Asp Phe Val 260 265 270 ata
gct act ggg gaa gtt cat agt gtc cgt gaa ttt gtt gag aaa tca 864 Ile
Ala Thr Gly Glu Val His Ser Val Arg Glu Phe Val Glu Lys Ser 275 280
285 ttc atg cac att gga aag acc att gtg tgg gaa gga aag aat gaa aat
912 Phe Met His Ile Gly Lys Thr Ile Val Trp Glu Gly Lys Asn Glu Asn
290 295 300 gaa gtg ggc aga tgt aaa gag acc ggc aaa att cat gtg act
gtg gat 960 Glu Val Gly Arg Cys Lys Glu Thr Gly Lys Ile His Val Thr
Val Asp 305 310 315 320 ctg aaa tac tac cga cca act gaa gtg gac ttc
ctg cag gga gac tgc 1008 Leu Lys Tyr Tyr Arg Pro Thr Glu Val Asp
Phe Leu Gln Gly Asp Cys 325 330 335 tcc aag gcg cag cag aaa ctg aac
tgg aag ccc cgc gtt gcc ttt gac 1056 Ser Lys Ala Gln Gln Lys Leu
Asn Trp Lys Pro Arg Val Ala Phe Asp 340 345 350 gag ctg gtg agg gag
atg gtg caa gcc gat gtg gag ctc atg aga acc 1104 Glu Leu Val Arg
Glu Met Val Gln Ala Asp Val Glu Leu Met Arg Thr 355 360 365 aac ccc
aac gcc tga gcacctctac aaaaaaattc gcgagacatg gactatggtg 1159 Asn
Pro Asn Ala 370 cagagccagc caaccagagt ccagccactc ctgagaccat
cgaccataaa ccctcgactg 1219 cctgtgtcgt ccccacagct aagagctggg
ccacaggttt gtgggcacca ggacggggac 1279 actccagagc taaggccact
tcgcttttgt caaaggctcc tctcaatgat tttgggaaat 1339 caagaagttt
aaaatcacat actcatttta cttgaaatta tgtcactaga caacttaaat 1399
ttttgagtct tgagattgtt tttctctttt cttattaaat gatctttcta tgacccagca
1459 aaaaaaaaaa aaaaaaggga tataaaaaaa aaaaaaaaaa aaaaa 1504 66 25
DNA Artificial Sequence Description of Artificial Sequence
Synthetic DNA 66 atgaagttgc actatggtga cctca 25 67 59 DNA
Cricetulus griseus 67 ccgacagcac ctgcctagta aaaatcatca atgaagtcaa
acctacagag atctacaat 59 68 25 DNA Artificial Sequence Description
of Artificial Sequence Synthetic DNA 68 gacttagcag agtacactgc agatg
25 69 25 DNA Artificial Sequence Description of Artificial Sequence
Synthetic DNA 69 accttggata gaaaggggtg gtctc 25 70 125 DNA
Cricetulus griseus 70 ttgatggagt tggcaccttg cggcttctgg atgcaattaa
gacttgtggc cttataaatt 60 ctgtgaagtt ctaccaggcc tcaactagtg
aactgtatgg aaaagtgcaa gaaatacccc 120 agaaa 125 71 372 PRT
Cricetulus griseus 71 Met Ala His Ala Pro Ala Ser Cys Pro Ser Ser
Arg Asn Ser Gly Asp 1 5 10 15 Gly Asp Lys Gly Lys Pro Arg Lys Val
Ala Leu Ile Thr Gly Ile Thr 20 25 30 Gly Gln Asp Gly Ser Tyr Leu
Ala Glu Phe Leu Leu Glu Lys Gly Tyr 35 40 45 Glu Val His Gly Ile
Val Arg Arg Ser Ser Ser Phe Asn Thr Gly Arg 50 55 60 Ile Glu His
Leu Tyr Lys Asn Pro Gln Ala His Ile Glu Gly Asn Met 65 70 75 80 Lys
Leu His Tyr Gly Asp Leu Thr Asp Ser Thr Cys Leu Val Lys Ile 85 90
95 Ile Asn Glu Val Lys Pro Thr Glu Ile Tyr Asn Leu Gly Ala Gln Ser
100 105 110 His Val Lys Ile Ser Phe Asp Leu Ala Glu Tyr Thr Ala Asp
Val Asp 115 120 125 Gly Val Gly Thr Leu Arg Leu Leu Asp Ala Ile Lys
Thr Cys Gly Leu 130 135 140 Ile Asn Ser Val Lys Phe Tyr Gln Ala Ser
Thr Ser Glu Leu Tyr Gly 145 150 155 160 Lys Val Gln Glu Ile Pro Gln
Lys Glu Thr Thr Pro Phe Tyr Pro Arg 165 170 175 Ser Pro Tyr Gly Ala
Ala Lys Leu Tyr Ala Tyr Trp Ile Val Val Asn 180 185 190 Phe Arg Glu
Ala Tyr Asn Leu Phe Ala Val Asn Gly Ile Leu Phe Asn 195 200 205 His
Glu Ser Pro Arg Arg Gly Ala Asn Phe Val Thr Arg Lys Ile Ser 210 215
220 Arg Ser Val Ala Lys Ile Tyr Leu Gly Gln Leu Glu Cys Phe Ser Leu
225 230 235 240 Gly Asn Leu Asp Ala Lys Arg Asp Trp Gly His Ala Lys
Asp Tyr Val 245 250 255 Glu Ala Met Trp Leu Met Leu Gln Asn Asp Glu
Pro Glu Asp Phe Val 260 265 270 Ile Ala Thr Gly Glu Val His Ser Val
Arg Glu Phe Val Glu Lys Ser 275 280 285 Phe Met His Ile Gly Lys Thr
Ile Val Trp Glu Gly Lys Asn Glu Asn 290 295 300 Glu Val Gly Arg Cys
Lys Glu Thr Gly Lys Ile His Val Thr Val Asp 305 310 315 320 Leu Lys
Tyr Tyr Arg Pro Thr Glu Val Asp Phe Leu Gln Gly Asp Cys 325 330 335
Ser Lys Ala Gln Gln Lys Leu Asn Trp Lys Pro Arg Val Ala Phe Asp 340
345 350 Glu Leu Val Arg Glu Met Val Gln Ala Asp Val Glu Leu Met Arg
Thr 355 360 365 Asn Pro Asn Ala 370 72 420 DNA Mus musculus 72 atg
gaa tgg atc tgg atc ttt ctc ttc ttc ctc tca gga act aca ggt 48 Met
Glu Trp Ile Trp Ile Phe Leu Phe Phe Leu Ser Gly Thr Thr Gly 1 5 10
15 gtc tac tcc cag gtt cag ctg cag cag tct gga gct gag gtg gcg agg
96 Val Tyr Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Ala Arg
20 25 30 ccc ggg gct tca gtg aaa ctg tcc tgc aag gct tct ggc tac
acc ttc 144 Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr
Thr Phe 35 40 45 act gac tac tat cta aac tgg gtg aag cag agg tct
gga cag ggc ctt 192 Thr Asp Tyr Tyr Leu Asn Trp Val Lys Gln Arg Ser
Gly Gln Gly Leu 50 55 60 gag tgg att gga gag att gat cct gga agt
gat agt ata tat tat aat 240 Glu Trp Ile Gly Glu Ile Asp Pro Gly Ser
Asp Ser Ile Tyr Tyr Asn 65 70 75 80 gaa aac ttg gag ggc agg gcc aca
ctg act gca gac aaa tcc tcc agc 288 Glu Asn Leu Glu Gly Arg Ala Thr
Leu Thr Ala Asp Lys Ser Ser Ser 85 90 95 aca gcc tac atg cag ctc
aac agc ctg aca tct gag gac tct gca gtc 336 Thr Ala Tyr Met Gln Leu
Asn Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110 tat ttc tgt gca
aga tat ggg tat tct aga tac gac gta agg ttt gtc 384 Tyr Phe Cys Ala
Arg Tyr Gly Tyr Ser Arg Tyr Asp Val Arg Phe Val 115 120 125 tac tgg
ggc caa ggg act ctg gtc act gtc tct aca 420 Tyr Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Thr 130 135 140 73 140 PRT Mus musculus 73 Met
Glu Trp Ile Trp Ile Phe Leu Phe Phe Leu Ser Gly Thr Thr Gly 1 5 10
15 Val Tyr Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Ala Arg
20 25 30 Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr
Thr Phe 35 40 45 Thr Asp Tyr Tyr Leu Asn Trp Val Lys Gln Arg Ser
Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Glu Ile Asp Pro Gly Ser
Asp Ser Ile Tyr Tyr Asn 65 70 75 80 Glu Asn Leu Glu Gly Arg Ala Thr
Leu Thr Ala Asp Lys Ser Ser Ser 85 90 95 Thr Ala Tyr Met Gln Leu
Asn Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110 Tyr Phe Cys Ala
Arg Tyr Gly Tyr Ser Arg Tyr Asp Val Arg Phe Val 115 120 125 Tyr Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Thr 130 135 140 74 393 DNA Mus
musculus 74 atg aag ttg cct gtt agg ctg ttg gtg ctg atg ttc tgg att
cct gct 48 Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile
Pro Ala 1 5 10 15 tcc agg agt gat gtt ttg atg acc caa act cca ctc
tcc ctg cct gtc 96 Ser Arg Ser Asp Val Leu Met Thr Gln Thr Pro Leu
Ser Leu Pro Val 20 25 30 agt ctt gga gat caa gcc tcc atc tct tgc
aga tct agt cag agt ctt 144 Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys
Arg Ser Ser Gln Ser Leu 35 40 45 gta cat agt aat gga aga acc tat
tta gaa tgg tac ctg cag aaa cct 192 Val His Ser Asn Gly Arg Thr Tyr
Leu Glu Trp Tyr Leu Gln Lys Pro 50 55 60 ggc cag tca cca aag gtc
ctg atc tac aaa gtt tcc aac cga att tct 240 Gly Gln Ser Pro Lys Val
Leu Ile Tyr Lys Val Ser Asn Arg Ile Ser 65 70 75 80 ggg gtc cca gac
agg ttc agt ggc agt gga tca ggg aca gat ttc aca 288 Gly Val Pro Asp
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 85 90 95 ctc aaa
atc agc aga gtg gag gct gag gat ctg gga gtt tat ttc tgc 336 Leu Lys
Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys 100 105 110
ttt cag ggt tca cat gtt ccg tac acg ttc gga ggg ggg acc aag ctg 384
Phe Gln Gly Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu 115
120 125 gaa ata aaa 393 Glu Ile Lys 130 75 131 PRT Mus musculus 75
Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro Ala 1 5
10 15 Ser Arg Ser Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro
Val 20 25 30 Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser
Gln Ser Leu 35 40 45 Val His Ser Asn Gly Arg Thr Tyr Leu Glu Trp
Tyr Leu Gln Lys Pro 50 55 60 Gly Gln Ser Pro Lys Val Leu Ile Tyr
Lys Val Ser Asn Arg Ile Ser 65 70 75 80 Gly Val Pro Asp Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr 85 90 95 Leu Lys Ile Ser Arg
Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys 100 105 110 Phe Gln Gly
Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu 115 120 125 Glu
Ile Lys 130 76 5 PRT Mus musculus 76 Asp Tyr Tyr Leu Asn 1 5 77 17
PRT Mus musculus 77 Glu Ile Asp Pro Gly Ser Asp Ser Ile Tyr Tyr Asn
Glu Asn Leu Glu 1 5 10 15 Gly 78 12 PRT Mus musculus 78 Tyr Gly Tyr
Ser Arg Tyr Asp Val Arg Phe Val Tyr 1 5 10 79 16 PRT Mus musculus
79 Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Arg Thr Tyr Leu Glu
1 5 10 15 80 7 PRT Mus musculus 80 Lys Val Ser Asn Arg Ile Ser 1 5
81 9 PRT Mus musculus 81 Phe Gln Gly Ser His Val Pro Tyr Thr 1 5 82
22 DNA Artificial Sequence Description of Artificial
Seque11cesynthetic DNA 82 ctgaattcgc ggccgctagt cc 22 83 39 DNA
Artificial Sequence Description of Artificial Sequence synthetic
DNA 83 atgggccctt ggtggaggct gtagagacag tgaccagag 39 84 22 DNA
Artificial Sequence Description of Artificial Sequencesynthetic DNA
84 ctgaattcgc ggccgctgct gt 22 85 28 DNA Artificial Sequence
Description of Artificial Sequence synthetic DNA 85 atcgtacgtt
ttatttccag cttggtcc 28 86 57 DNA Artificial Sequence Description of
Artificial Sequencesynthetic DNA 86 ttgttggtac cgaattcttt
cagggccccg gagccccgag agcccaaatc ttgtgac 57 87 31 DNA Artificial
Sequence Description of Artificial Sequence synthetic DNA 87
gcgaattcca ccatgggcag cccccgctcc g 31 88 32 DNA Artificial Sequence
Description of Artificial Sequencesynthetic DNA 88 cgggatccct
atcggggctc cggggcccaa gt 32 89 1341 DNA Artificial Sequence
Description of Artificial Sequencesynthetic DNA 89 atg ggc agc ccc
cgc tcc gcg ctg agc tgc ctg ctg ttg cac ttg ctg 48 Met Gly Ser Pro
Arg Ser Ala Leu Ser Cys Leu Leu Leu His Leu Leu 1 5 10 15 gtt ctc
tgc ctc caa gcc cag gta act gtt cag tcc tca cct aat ttt 96 Val Leu
Cys Leu Gln Ala Gln Val Thr Val Gln Ser Ser Pro Asn Phe 20 25 30
aca cag cat gtg agg gag cag agc ctg gtg acg gat cag ctc agc cgc 144
Thr Gln His Val Arg Glu Gln Ser Leu Val Thr Asp Gln Leu Ser Arg 35
40 45 cgc ctc atc cgg acc tac cag ctc tac agc cgc acc agc ggg aag
cac 192 Arg Leu Ile Arg Thr Tyr Gln Leu Tyr Ser Arg Thr Ser Gly Lys
His 50 55 60 gtg cag gtc ctg gcc aac aag cgc atc aac gcc atg gca
gaa gac gga 240 Val Gln Val Leu Ala Asn Lys Arg Ile Asn Ala Met Ala
Glu Asp Gly 65 70 75 80 gac ccc ttc gcg aag ctc att gtg gag acc gat
act ttt gga agc aga 288 Asp Pro Phe Ala Lys Leu Ile Val Glu Thr Asp
Thr Phe Gly Ser Arg 85 90 95 gtc cga gtt cgc ggc gca gag aca ggt
ctc tac atc tgc atg aac aag 336 Val Arg Val Arg Gly Ala Glu Thr Gly
Leu Tyr Ile Cys Met Asn Lys 100 105 110 aag ggg aag cta att gcc aag
agc aac ggc aaa ggc aag gac tgc gta 384 Lys Gly Lys Leu Ile Ala Lys
Ser Asn Gly Lys Gly Lys Asp Cys Val 115 120 125 ttc aca gag atc gtg
ctg gag aac aac tac acg gcg ctg cag aac gcc 432 Phe Thr Glu Ile Val
Leu Glu Asn Asn Tyr Thr Ala Leu Gln Asn Ala 130 135 140 aag tac gag
ggc tgg tac atg gcc ttt acc cgc aag ggc cgg ccc cgc
480 Lys Tyr Glu Gly Trp Tyr Met Ala Phe Thr Arg Lys Gly Arg Pro Arg
145 150 155 160 aag ggc tcc aag acg cgc cag cat cag cgc gag gtg cac
ttc atg aag 528 Lys Gly Ser Lys Thr Arg Gln His Gln Arg Glu Val His
Phe Met Lys 165 170 175 cgc ctg ccg cgg ggc cac cac acc acc gag cag
agc ctg cgc ttc gag 576 Arg Leu Pro Arg Gly His His Thr Thr Glu Gln
Ser Leu Arg Phe Glu 180 185 190 ttc ctc aac tac ccg ccc ttc acg cgc
agc ctg cgc ggc agc cag agg 624 Phe Leu Asn Tyr Pro Pro Phe Thr Arg
Ser Leu Arg Gly Ser Gln Arg 195 200 205 act tgg gcc ccg gag ccc cga
gag ccc aaa tct tgt gac aaa act cac 672 Thr Trp Ala Pro Glu Pro Arg
Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 aca tgc cca ccg tgc
cca gca cct gaa ctc ctg ggg gga ccg tca gtc 720 Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 ttc ctc
ttc ccc cca aaa ccc aag gac acc ctc atg atc tcc cgg acc 768 Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255
cct gag gtc aca tgc gtg gtg gtg gac gtg agc cac gaa gac cct gag 816
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260
265 270 gtc aag ttc aac tgg tac gtg gac ggc gtg gag gtg cat aat gcc
aag 864 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys 275 280 285 aca aag ccg cgg gag gag cag tac aac agc acg tac cgt
gtg gtc agc 912 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
Val Val Ser 290 295 300 gtc ctc acc gtc ctg cac cag gac tgg ctg aat
ggc aag gag tac aag 960 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys 305 310 315 320 tgc aag gtc tcc aac aaa gcc ctc
cca gcc ccc atc gag aaa acc atc 1008 Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 tcc aaa gcc aaa ggg
cag ccc cga gaa cca cag gtg tac acc ctg ccc 1056 Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 cca tcc
cgg gat gag ctg acc aag aac cag gtc agc ctg acc tgc ctg 1104 Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360
365 gtc aaa ggc ttc tat ccc agc gac atc gcc gtg gag tgg gag agc aat
1152 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn 370 375 380 ggg cag ccg gag aac aac tac aag acc acg cct ccc gtg
ctg gac tcc 1200 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser 385 390 395 400 gac ggc tcc ttc ttc ctc tac agc aag
ctc acc gtg gac aag agc agg 1248 Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 tgg cag cag ggg aac gtc
ttc tca tgc tcc gtg atg cat gag gct ctg 1296 Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 cac aac cac
tac acg cag aag agc ctc tcc ctg tct ccg ggt aaa 1341 His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 90 447
PRT homo sapiens 90 Met Gly Ser Pro Arg Ser Ala Leu Ser Cys Leu Leu
Leu His Leu Leu 1 5 10 15 Val Leu Cys Leu Gln Ala Gln Val Thr Val
Gln Ser Ser Pro Asn Phe 20 25 30 Thr Gln His Val Arg Glu Gln Ser
Leu Val Thr Asp Gln Leu Ser Arg 35 40 45 Arg Leu Ile Arg Thr Tyr
Gln Leu Tyr Ser Arg Thr Ser Gly Lys His 50 55 60 Val Gln Val Leu
Ala Asn Lys Arg Ile Asn Ala Met Ala Glu Asp Gly 65 70 75 80 Asp Pro
Phe Ala Lys Leu Ile Val Glu Thr Asp Thr Phe Gly Ser Arg 85 90 95
Val Arg Val Arg Gly Ala Glu Thr Gly Leu Tyr Ile Cys Met Asn Lys 100
105 110 Lys Gly Lys Leu Ile Ala Lys Ser Asn Gly Lys Gly Lys Asp Cys
Val 115 120 125 Phe Thr Glu Ile Val Leu Glu Asn Asn Tyr Thr Ala Leu
Gln Asn Ala 130 135 140 Lys Tyr Glu Gly Trp Tyr Met Ala Phe Thr Arg
Lys Gly Arg Pro Arg 145 150 155 160 Lys Gly Ser Lys Thr Arg Gln His
Gln Arg Glu Val His Phe Met Lys 165 170 175 Arg Leu Pro Arg Gly His
His Thr Thr Glu Gln Ser Leu Arg Phe Glu 180 185 190 Phe Leu Asn Tyr
Pro Pro Phe Thr Arg Ser Leu Arg Gly Ser Gln Arg 195 200 205 Thr Trp
Ala Pro Glu Pro Arg Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225
230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Gln Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345
350 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 91 1245 DNA
Cricetulus griseus 91 gaacttcacc caagccatgt gacaattgaa ggctgtaccc
ccagacccta acatcttgga 60 gccctgtaga ccagggagtg cttctggccg
tggggtgacc tagctcttct accaccatga 120 acagggcccc tctgaagcgg
tccaggatcc tgcgcatggc gctgactgga ggctccactg 180 cctctgagga
ggcagatgaa gacagcagga acaagccgtt tctgctgcgg gcgctgcaga 240
tcgcgctggt cgtctctctc tactgggtca cctccatctc catggtattc ctcaacaagt
300 acctgctgga cagcccctcc ctgcagctgg atacccctat cttcgtcact
ttctaccaat 360 gcctggtgac ctctctgctg tgcaagggcc tcagcactct
ggccacctgc tgccctggca 420 ccgttgactt ccccaccctg aacctggacc
ttaaggtggc ccgcagcgtg ctgccactgt 480 cggtagtctt cattggcatg
ataagtttca ataacctctg cctcaagtac gtaggggtgg 540 ccttctacaa
cgtggggcgc tcgctcacca ccgtgttcaa tgtgcttctg tcctacctgc 600
tgctcaaaca gaccacttcc ttctatgccc tgctcacatg tggcatcatc attggtggtt
660 tctggctggg tatagaccaa gagggagctg agggcaccct gtccctcata
ggcaccatct 720 tcggggtgct ggccagcctc tgcgtctccc tcaatgccat
ctataccaag aaggtgctcc 780 cagcagtgga caacagcatc tggcgcctaa
ccttctataa caatgtcaat gcctgtgtgc 840 tcttcttgcc cctgatggtt
ctgctgggtg agctccgtgc cctccttgac tttgctcatc 900 tgtacagtgc
ccacttctgg ctcatgatga cgctgggtgg cctcttcggc tttgccattg 960
gctatgtgac aggactgcag atcaaattca ccagtcccct gacccacaat gtatcaggca
1020 cagccaaggc ctgtgcgcag acagtgctgg ccgtgctcta ctatgaagag
actaagagct 1080 tcctgtggtg gacaagcaac ctgatggtgc tgggtggctc
ctcagcctat acctgggtca 1140 ggggctggga gatgcagaag acccaagagg
accccagctc caaagagggt gagaagagtg 1200 ctattggggt gtgagcttct
tcagggacct gggactgaac ccaag 1245 92 365 PRT Cricetulus griseus 92
Met Asn Arg Ala Pro Leu Lys Arg Ser Arg Ile Leu Arg Met Ala Leu 1 5
10 15 Thr Gly Gly Ser Thr Ala Ser Glu Glu Ala Asp Glu Asp Ser Arg
Asn 20 25 30 Lys Pro Phe Leu Leu Arg Ala Leu Gln Ile Ala Leu Val
Val Ser Leu 35 40 45 Tyr Trp Val Thr Ser Ile Ser Met Val Phe Leu
Asn Lys Tyr Leu Leu 50 55 60 Asp Ser Pro Ser Leu Gln Leu Asp Thr
Pro Ile Phe Val Thr Phe Tyr 65 70 75 80 Gln Cys Leu Val Thr Ser Leu
Leu Cys Lys Gly Leu Ser Thr Leu Ala 85 90 95 Thr Cys Cys Pro Gly
Thr Val Asp Phe Pro Thr Leu Asn Leu Asp Leu 100 105 110 Lys Val Ala
Arg Ser Val Leu Pro Leu Ser Val Val Phe Ile Gly Met 115 120 125 Ile
Ser Phe Asn Asn Leu Cys Leu Lys Tyr Val Gly Val Ala Phe Tyr 130 135
140 Asn Val Gly Arg Ser Leu Thr Thr Val Phe Asn Val Leu Leu Ser Tyr
145 150 155 160 Leu Leu Leu Lys Gln Thr Thr Ser Phe Tyr Ala Leu Leu
Thr Cys Gly 165 170 175 Ile Ile Ile Gly Gly Phe Trp Leu Gly Ile Asp
Gln Glu Gly Ala Glu 180 185 190 Gly Thr Leu Ser Leu Ile Gly Thr Ile
Phe Gly Val Leu Ala Ser Leu 195 200 205 Cys Val Ser Leu Asn Ala Ile
Tyr Thr Lys Lys Val Leu Pro Ala Val 210 215 220 Asp Asn Ser Ile Trp
Arg Leu Thr Phe Tyr Asn Asn Val Asn Ala Cys 225 230 235 240 Val Leu
Phe Leu Pro Leu Met Val Leu Leu Gly Glu Leu Arg Ala Leu 245 250 255
Leu Asp Phe Ala His Leu Tyr Ser Ala His Phe Trp Leu Met Met Thr 260
265 270 Leu Gly Gly Leu Phe Gly Phe Ala Ile Gly Tyr Val Thr Gly Leu
Gln 275 280 285 Ile Lys Phe Thr Ser Pro Leu Thr His Asn Val Ser Gly
Thr Ala Lys 290 295 300 Ala Cys Ala Gln Thr Val Leu Ala Val Leu Tyr
Tyr Glu Glu Thr Lys 305 310 315 320 Ser Phe Leu Trp Trp Thr Ser Asn
Leu Met Val Leu Gly Gly Ser Ser 325 330 335 Ala Tyr Thr Trp Val Arg
Gly Trp Glu Met Gln Lys Thr Gln Glu Asp 340 345 350 Pro Ser Ser Lys
Glu Gly Glu Lys Ser Ala Ile Gly Val 355 360 365 93 1095 DNA Homo
sapiens 93 atgaataggg cccctctgaa gcggtccagg atcctgcaca tggcgctgac
cggggcctca 60 gacccctctg cagaggcaga ggccaacggg gagaagccct
ttctgctgcg ggcattgcag 120 atcgcgctgg tggtctccct ctactgggtc
acctccatct ccatggtgtt ccttaataag 180 tacctgctgg acagcccctc
cctgcggctg gacaccccca tcttcgtcac cttctaccag 240 tgcctggtga
ccacgctgct gtgcaaaggc ctcagcgctc tggccgcctg ctgccctggt 300
gccgtggact tccccagctt gcgcctggac ctcagggtgg cccgcagcgt cctgcccctg
360 tcggtggtct tcatcggcat gatcaccttc aataacctct gcctcaagta
cgtcggtgtg 420 gccttctaca atgtgggccg ctcactcacc accgtcttca
acgtgctgct ctcctacctg 480 ctgctcaagc agaccacctc cttctatgcc
ctgctcacct gcggtatcat catcgggggc 540 ttctggcttg gtgtggacca
ggagggggca gaaggcaccc tgtcgtggct gggcaccgtc 600 ttcggcgtgc
tggctagcct ctgtgtctcg ctcaacgcca tctacaccac gaaggtgctc 660
ccggcggtgg acggcagcat ctggcgcctg actttctaca acaacgtcaa cgcctgcatc
720 ctcttcctgc ccctgctcct gctgctcggg gagcttcagg ccctgcgtga
ctttgcccag 780 ctgggcagtg cccacttctg ggggatgatg acgctgggcg
gcctgtttgg ctttgccatc 840 ggctacgtga caggactgca gatcaagttc
accagtccgc tgacccacaa tgtgtcgggc 900 acggccaagg cctgtgccca
gacagtgctg gccgtgctct actacgagga gaccaagagc 960 ttcctctggt
ggacgagcaa catgatggtg ctgggcggct cctccgccta cacctgggtc 1020
aggggctggg agatgaagaa gactccggag gagcccagcc ccaaagacag cgagaagagc
1080 gccatggggg tgtga 1095 94 364 PRT Homo sapiens 94 Met Asn Arg
Ala Pro Leu Lys Arg Ser Arg Ile Leu His Met Ala Leu 1 5 10 15 Thr
Gly Ala Ser Asp Pro Ser Ala Glu Ala Glu Ala Asn Gly Glu Lys 20 25
30 Pro Phe Leu Leu Arg Ala Leu Gln Ile Ala Leu Val Val Ser Leu Tyr
35 40 45 Trp Val Thr Ser Ile Ser Met Val Phe Leu Asn Lys Tyr Leu
Leu Asp 50 55 60 Ser Pro Ser Leu Arg Leu Asp Thr Pro Ile Phe Val
Thr Phe Tyr Gln 65 70 75 80 Cys Leu Val Thr Thr Leu Leu Cys Lys Gly
Leu Ser Ala Leu Ala Ala 85 90 95 Cys Cys Pro Gly Ala Val Asp Phe
Pro Ser Leu Arg Leu Asp Leu Arg 100 105 110 Val Ala Arg Ser Val Leu
Pro Leu Ser Val Val Phe Ile Gly Met Ile 115 120 125 Thr Phe Asn Asn
Leu Cys Leu Lys Tyr Val Gly Val Ala Phe Tyr Asn 130 135 140 Val Gly
Arg Ser Leu Thr Thr Val Phe Asn Val Leu Leu Ser Tyr Leu 145 150 155
160 Leu Leu Lys Gln Thr Thr Ser Phe Tyr Ala Leu Leu Thr Cys Gly Ile
165 170 175 Ile Ile Gly Gly Phe Trp Leu Gly Val Asp Gln Glu Gly Ala
Glu Gly 180 185 190 Thr Leu Ser Trp Leu Gly Thr Val Phe Gly Val Leu
Ala Ser Leu Cys 195 200 205 Val Ser Leu Asn Ala Ile Tyr Thr Thr Lys
Val Leu Pro Ala Val Asp 210 215 220 Gly Ser Ile Trp Arg Leu Thr Phe
Tyr Asn Asn Val Asn Ala Cys Ile 225 230 235 240 Leu Phe Leu Pro Leu
Leu Leu Leu Leu Gly Glu Leu Gln Ala Leu Arg 245 250 255 Asp Phe Ala
Gln Leu Gly Ser Ala His Phe Trp Gly Met Met Thr Leu 260 265 270 Gly
Gly Leu Phe Gly Phe Ala Ile Gly Tyr Val Thr Gly Leu Gln Ile 275 280
285 Lys Phe Thr Ser Pro Leu Thr His Asn Val Ser Gly Thr Ala Lys Ala
290 295 300 Cys Ala Gln Thr Val Leu Ala Val Leu Tyr Tyr Glu Glu Thr
Lys Ser 305 310 315 320 Phe Leu Trp Trp Thr Ser Asn Met Met Val Leu
Gly Gly Ser Ser Ala 325 330 335 Tyr Thr Trp Val Arg Gly Trp Glu Met
Lys Lys Thr Pro Glu Glu Pro 340 345 350 Ser Pro Lys Asp Ser Glu Lys
Ser Ala Met Gly Val 355 360 95 2609 DNA Mus musculus 95 gagccgaggg
tggtgctgca ggtgcacccg agggcaccgc cgagggtgag caccaggtcc 60
ctgcatcagc caggacacca gagcccagtc gggtggacgg acgggcgcct ctgaagcggt
120 ccaggatcct gcgcatggcg ctgactggag tctctgctgt ctccgaggag
tcagagagcg 180 ggaacaagcc atttctgctc cgggctctgc agatcgcgct
ggtggtctct ctctactggg 240 tcacctccat ttccatggta ttcctcaaca
agtacctgct ggacagcccc tccctgcagc 300 tggatacccc catttttgtc
accttctacc aatgcctggt gacctcactg ctgtgcaagg 360 gcctcagcac
tctggccacc tgctgccccg gcatggtaga cttccccacc ctaaacctgg 420
acctcaaggt ggcccgaagt gtgctgccgc tgtcagtggt ctttatcggc atgataacct
480 tcaataacct ctgcctcaag tacgtagggg tgcccttcta caacgtggga
cgctcgctca 540 ccaccgtgtt caacgttctt ctctcctacc tgctgctcaa
acagaccact tccttctatg 600 ccctgctcac ctgcggcgtc atcattggtg
gtttctggct gggtatagac caagaaggag 660 ctgagggaac cttgtccctg
acgggcacca tcttcggggt gctggccagc ctctgcgtct 720 ccctcaatgc
catctatacc aagaaggtgc tccctgcagt agaccacagt atctggcgcc 780
taaccttcta taacaatgtc aatgcctgcg tgctcttctt gcccctgatg atagtgctgg
840 gcgagctccg tgccctcctg gccttcactc atctgagcag tgcccacttc
tggctcatga 900 tgacgctggg tggcctgttt ggctttgcca tcggctatgt
gacaggactg cagatcaaat 960 tcaccagtcc cctgacccat aacgtgtcag
gcacggccaa ggcctgtgca cagacagtgc 1020 tggccgtgct ctactacgaa
gagattaaga gcttcctgtg gtggacaagc aacctgatgg 1080 tgctgggtgg
ctcctccgcc tacacctggg tcaggggctg ggagatgcag aagacccagg 1140
aggaccccag ctccaaagat ggtgagaaga gtgctatcag ggtgtgagct ccttcaggga
1200 gccagggctg agctcgggtg gggcctgccc agcacggaag gcttcccata
gagcctactg 1260 ggtatggccc tgagcaataa tgtttacatc cttctcagaa
gaccatctaa gaagagccag 1320 gttctttcct gataatgtca gaaagctgcc
aaatctcctg cctgccccat cttctagtct 1380 tgggaaagcc ctaccaggag
tggcaccctt cctgcctcct cctggggcct gtctacctcc 1440 atatggtctc
tggggttggg gccagctgca ctctttgggc actggactga tgaagtgatg 1500
tcttactttc tacacaaggg agatgggttg tgaccctact atagctagtt gaagggagct
1560 gtgtaacccc acatctctgg ggccctgggc aggtagcata atagctaggt
gctattaaca 1620 tcaataacac ttcagactac ctttggaggc agttgggagc
tgagccgaga gagagagatg 1680 gccattctgc cctcttctgt gtggatgggt
atgacagacc aactgtccat ggggtgactg 1740 acacctccac acttcatatt
ttcaacttta gaaaaggggg agccacacgt tttacagatt 1800 aagtggagtg
atgaatgcct ctacagcccc taaccccact ttccctgcct ggcttctctt 1860
ggcccagaag ggccaccatc ctgttctcca acacctgacc cagctatctg gctatactct
1920 ctttctgtac tcccttcccc ttcccccccc cattagcctc ctccccaaca
cctccatctt 1980 caggcaggaa gtggggtcca ctcagcctct gttcccatct
gcttggaccc ctgagcctct 2040 catgaaggta ggcttatgtt ctctgaggct
ggggccggag gagcgcactg attctcggag 2100 ttatcccatc aggctcctgt
cacaaaatag cctaggccgt gtgtctaaga acagtggagg 2160 ttggcttata
actgttctgg gggcagcgaa gcccacatca aggtactcat agacccagta 2220
tttctgagga aacccctgtc cacatcctca cttggtaaag gcacagataa tctccctcag
2280 gcctcttgta taggagcact agccctggga
gggctccgcc ccatgacctg atcaccccaa 2340 agccttcaac agaaggattc
caacatgaat ttggggacag aagcactcag accacgatgc 2400 ccagcaccac
accctcctat cctcagggta gctgtcactg tcctagtccc ttctgtttgg 2460
ccttttgtac cctcatttcc ttggcgtcat gtttgatgtc tttgtctctt tcgtgagaag
2520 atggggaaac catgtcagcc tctgcttccg acttcccatg ggttctaatg
aagttggtgg 2580 ggcctgatgc cctgagttgt atgtgattt 2609 96 350 PRT Mus
musculus 96 Met Ala Leu Thr Gly Val Ser Ala Val Ser Glu Glu Ser Glu
Ser Gly 1 5 10 15 Asn Lys Pro Phe Leu Leu Arg Ala Leu Gln Ile Ala
Leu Val Val Ser 20 25 30 Leu Tyr Trp Val Thr Ser Ile Ser Met Val
Phe Leu Asn Lys Tyr Leu 35 40 45 Leu Asp Ser Pro Ser Leu Gln Leu
Asp Thr Pro Ile Phe Val Thr Phe 50 55 60 Tyr Gln Cys Leu Val Thr
Ser Leu Leu Cys Lys Gly Leu Ser Thr Leu 65 70 75 80 Ala Thr Cys Cys
Pro Gly Met Val Asp Phe Pro Thr Leu Asn Leu Asp 85 90 95 Leu Lys
Val Ala Arg Ser Val Leu Pro Leu Ser Val Val Phe Ile Gly 100 105 110
Met Ile Thr Phe Asn Asn Leu Cys Leu Lys Tyr Val Gly Val Pro Phe 115
120 125 Tyr Asn Val Gly Arg Ser Leu Thr Thr Val Phe Asn Val Leu Leu
Ser 130 135 140 Tyr Leu Leu Leu Lys Gln Thr Thr Ser Phe Tyr Ala Leu
Leu Thr Cys 145 150 155 160 Gly Val Ile Ile Gly Gly Phe Trp Leu Gly
Ile Asp Gln Glu Gly Ala 165 170 175 Glu Gly Thr Leu Ser Leu Thr Gly
Thr Ile Phe Gly Val Leu Ala Ser 180 185 190 Leu Cys Val Ser Leu Asn
Ala Ile Tyr Thr Lys Lys Val Leu Pro Ala 195 200 205 Val Asp His Ser
Ile Trp Arg Leu Thr Phe Tyr Asn Asn Val Asn Ala 210 215 220 Cys Val
Leu Phe Leu Pro Leu Met Ile Val Leu Gly Glu Leu Arg Ala 225 230 235
240 Leu Leu Ala Phe Thr His Leu Ser Ser Ala His Phe Trp Leu Met Met
245 250 255 Thr Leu Gly Gly Leu Phe Gly Phe Ala Ile Gly Tyr Val Thr
Gly Leu 260 265 270 Gln Ile Lys Phe Thr Ser Pro Leu Thr His Asn Val
Ser Gly Thr Ala 275 280 285 Lys Ala Cys Ala Gln Thr Val Leu Ala Val
Leu Tyr Tyr Glu Glu Ile 290 295 300 Lys Ser Phe Leu Trp Trp Thr Ser
Asn Leu Met Val Leu Gly Gly Ser 305 310 315 320 Ser Ala Tyr Thr Trp
Val Arg Gly Trp Glu Met Gln Lys Thr Gln Glu 325 330 335 Asp Pro Ser
Ser Lys Asp Gly Glu Lys Ser Ala Ile Arg Val 340 345 350 97 1053 DNA
Rattus norvegiucus 97 atggcgctga ctggagcctc tgctgtctct gaggaggcag
acagcgagaa caagccattt 60 ctgctacggg ctctgcagat cgcgctggtg
gtttctctct actgggtcac ctccatctcc 120 atggtattcc tcaacaagta
cctgctggac agcccctccc tgcagctgga tacccccatc 180 ttcgtcacct
tctaccaatg cctggtgacc tcactgctgt gcaagggcct cagcactctg 240
gccacctgct gccctggcat ggtagacttc cccaccctaa acctggacct caaggtggcc
300 cgaagtgtgc tgccgctgtc cgtggtcttt atcggcatga taaccttcaa
taacctctgc 360 ctcaagtacg tgggggtggc cttctacaac gtgggacgct
cgctcactac cgtgttcaat 420 gtgcttctct cctacctgct gcttaaacag
accacttcct tttatgccct gctcacctgt 480 gccatcatca ttggtggttt
ctggctggga atagatcaag agggagctga gggcaccctg 540 tccctgacgg
gcaccatctt cggggtgctg gccagcctct gtgtctcact caatgccatc 600
tacaccaaga aggtgctccc tgccgtagac cacagtatct ggcgcctaac cttctataac
660 aacgtcaacg cctgtgtgct cttcttgccc ctgatggtag tgctgggcga
gctccatgct 720 ctcctggcct tcgctcatct gaacagcgcc cacttctggg
tcatgatgac gctgggtgga 780 ctcttcggct ttgccattgg ctatgtgaca
ggactgcaga tcaaattcac cagtcccctg 840 acccataatg tgtcgggcac
agccaaggcc tgtgcacaga cagtgctggc tgtgctctac 900 tatgaagaga
ttaagagctt cctgtggtgg acaagcaact tgatggtgct gggtggctcc 960
tctgcctaca cctgggtcag gggctgggag atgcagaaga cccaggagga ccccagctcc
1020 aaagagggtg agaagagtgc tatcggggtg tga 1053 98 350 PRT Rattus
norvegiucus 98 Met Ala Leu Thr Gly Ala Ser Ala Val Ser Glu Glu Ala
Asp Ser Glu 1 5 10 15 Asn Lys Pro Phe Leu Leu Arg Ala Leu Gln Ile
Ala Leu Val Val Ser 20 25 30 Leu Tyr Trp Val Thr Ser Ile Ser Met
Val Phe Leu Asn Lys Tyr Leu 35 40 45 Leu Asp Ser Pro Ser Leu Gln
Leu Asp Thr Pro Ile Phe Val Thr Phe 50 55 60 Tyr Gln Cys Leu Val
Thr Ser Leu Leu Cys Lys Gly Leu Ser Thr Leu 65 70 75 80 Ala Thr Cys
Cys Pro Gly Met Val Asp Phe Pro Thr Leu Asn Leu Asp 85 90 95 Leu
Lys Val Ala Arg Ser Val Leu Pro Leu Ser Val Val Phe Ile Gly 100 105
110 Met Ile Thr Phe Asn Asn Leu Cys Leu Lys Tyr Val Gly Val Ala Phe
115 120 125 Tyr Asn Val Gly Arg Ser Leu Thr Thr Val Phe Asn Val Leu
Leu Ser 130 135 140 Tyr Leu Leu Leu Lys Gln Thr Thr Ser Phe Tyr Ala
Leu Leu Thr Cys 145 150 155 160 Ala Ile Ile Ile Gly Gly Phe Trp Leu
Gly Ile Asp Gln Glu Gly Ala 165 170 175 Glu Gly Thr Leu Ser Leu Thr
Gly Thr Ile Phe Gly Val Leu Ala Ser 180 185 190 Leu Cys Val Ser Leu
Asn Ala Ile Tyr Thr Lys Lys Val Leu Pro Ala 195 200 205 Val Asp His
Ser Ile Trp Arg Leu Thr Phe Tyr Asn Asn Val Asn Ala 210 215 220 Cys
Val Leu Phe Leu Pro Leu Met Val Val Leu Gly Glu Leu His Ala 225 230
235 240 Leu Leu Ala Phe Ala His Leu Asn Ser Ala His Phe Trp Val Met
Met 245 250 255 Thr Leu Gly Gly Leu Phe Gly Phe Ala Ile Gly Tyr Val
Thr Gly Leu 260 265 270 Gln Ile Lys Phe Thr Ser Pro Leu Thr His Asn
Val Ser Gly Thr Ala 275 280 285 Lys Ala Cys Ala Gln Thr Val Leu Ala
Val Leu Tyr Tyr Glu Glu Ile 290 295 300 Lys Ser Phe Leu Trp Trp Thr
Ser Asn Leu Met Val Leu Gly Gly Ser 305 310 315 320 Ser Ala Tyr Thr
Trp Val Arg Gly Trp Glu Met Gln Lys Thr Gln Glu 325 330 335 Asp Pro
Ser Ser Lys Glu Gly Glu Lys Ser Ala Ile Gly Val 340 345 350 99 7752
DNA Mus musculus 99 agcgttgcaa gttcagccga gggtggtgct gcaggtgcac
ccgagggcac cgccgagggt 60 gagcaccagg tccctgcatc agccaggaca
ccagagccca gtcgggtgga cggacggtac 120 gttctggaag ggaaagggcc
ccgggaaggg gatacagcat tgagaagctc agaggctttg 180 gtctgggcat
ccagaatgga ctttatctgg aggaggtgac atggcttctc gcctctggaa 240
gtgctgcttg gatctccggg acttcatgtc ctgactaggt ctggaagcgg tgaaaatagg
300 ggtaggaaaa aaggagagga ctgcaacaag gtcttcccga gtggcctgag
ctcgagggac 360 gagggaggtg caacggtggg gagccgggcg caagggctgg
gcggagggag ggggggggtc 420 tccctaagca gaaaggtggt attccatttt
ctgggtagat ggtgaagatg cacctgaccg 480 agtctggtcg atctgaagat
atcaggggaa aagatagtgc gggtggaggg gagaatgaca 540 gaaccttcca
gaaaatggga gaggctatag cacttgcaaa cccttccctg atctccgggg 600
actcccggaa gaagagggca ggtctgtggg cataggtgca gacttgccgg ggagctcttg
660 acggccgcgg gaagtggcaa cggcctgcga gctggccctt taaggcggct
cgtaggcgtg 720 tcaggaaatg cgcgcagggc ccgccctgct cggtaagtgg
cccgggaccc gcgtcgctga 780 gccggaactt gaattcggct cgtggcaacc
gcagggcctt gctccggtca ggcccctgtc 840 cgtgtccctc gagacgcctt
cctgagcctc ggtgatctcc ctgcagcacg ccctcctttc 900 ggctctgcgg
gtgcttccgg gggttcccgc agcccatgct tcccacgcgg tccgcgtcca 960
gttatttcct cctccgctcc gtccttcctt cgctctctcg cttcctttct ccctgcgact
1020 cacgtgtccc ctgtcctcaa actggccatg gctgtcaaag cccacatcct
tagttaggcc 1080 ccttctccct tccctgggtc ttgtttcgtg acaccacctc
cctcccccgc cccgggagcg 1140 agcaagatga ggagcggtgc acctcggcaa
atccggaagc agaacttcat ccaagaagga 1200 ggggaccgat aggtcatccc
atgtgacagt tgaaggctgc agccacagac cctagctgct 1260 tgaagccctg
tagtccaggg actgcttctg gccgtaaggt gacccagctc ttctgccacc 1320
atgaacaggg cgcctctgaa gcggtccagg atcctgcgca tggcgctgac tggagtctct
1380 gctgtctccg aggagtcaga gagcgggaac aagccatttc tgctccgggc
tctgcagatc 1440 gcgctggtgg tctctctcta ctgggtcacc tccatttcca
tggtattcct caacaagtac 1500 ctgctggaca gcccctccct gcagctggat
acccccattt ttgtcacctt ctaccaatgc 1560 ctggtgacct cactgctgtg
caagggcctc agcactctgg ccacctgctg ccccggcatg 1620 gtagacttcc
ccaccctaaa cctggacctc aaggtggccc gaagtgtgct gccgctgtca 1680
gtggtcttta tcggcatgat aaccttcaat aacctctgcc tcaagtacgt aggggtgccc
1740 ttctacaacg tgggacgctc gctcaccacc gtgttcaacg ttcttctctc
ctacctgctg 1800 ctcaaacaga ccacttcctt ctatgccctg ctcacctgcg
gcgtcatcat tggtgagtgg 1860 gactgggggc gtggggagca ggaatcgtaa
agatcaatac cacattactc attatctgtc 1920 ccaggtcttt tgcaccacca
gtcataggga gagacctgta gagaacaaat aacttcctta 1980 ctgtgactca
gtaagttagg gatccagcca aggtgaacat aataatgtta ggcagacact 2040
acagcaaagc cagccagaca ctcagatcta gctaagcatt tgagccatgt taatgtaacg
2100 gatccccatt acaaggtata atatagctgc gttttatgga gagaaaccca
aggcacagag 2160 aagctaagta gctgggatca cacaggtaat cactgacgta
gcagaaattt gcacataagc 2220 agttacctcc ataggttaca ctcttgacca
acacaccact gttctcaaga ggtcaagggt 2280 gaactcaggt catcacaatt
ggcacaagta cctctaccca ctgagccatt tcagtggtcc 2340 agtcaatatg
tgtgtgcttt aagaggcttt aactaccttc tcagatgtga ccataagtaa 2400
ttaattaccg ataggagcgt tgtgctgatc attacacttg tagcatcctc tttattgtac
2460 ccataagctc tctgagtggc ggcatctctg tgaaactgca gctcggagag
gctgcgctcc 2520 ttgccacagc cccacaacta agaagcagat agtctgggac
gcagtcccca gttggtcata 2580 ctccctggcc tgtgtttcaa gccagtctgc
tttgctcctg acccttggga gttagcgcaa 2640 tgaaaaccaa cactatcact
acagtctaaa tgtgctttta aatgaaagcc caggaacttt 2700 gaagcatccg
gccccttaac ggcagccact atgtcctgat tccgccaaca tcttttcagt 2760
gcccggcagt cacatggagc aagggcctct tggcttggac agcatgtgtt agggaacatg
2820 tttgccactt tgaatgaatt tagtggctgc tgggttacag agaccagggc
atctttcccc 2880 tcagagtcct gaatgaacga aaagcaacct tcatttgtac
ctgctctgga ttttagttcg 2940 tcttgtttgg cctatttaga tgtccctggt
gtctctgagg cccaggctgg gtgctctaga 3000 tgtagggacc aggccaacct
gtactgtctt ccctagaaac attgccctgg ttgggcagct 3060 cctggatcca
gggttaaggg gtctgggcgg agagaggtca gatagtggca ggatgcctcc 3120
cactgccccc acatacatac cctaagagat ctggtactcc tccttccagc ctacaagcta
3180 ccgtggggtc ccacttcagt ccaccagccc tgccaacgtt agaggggatg
ggcctcctag 3240 taggagaact tacatgcagg aaggtacagt ctctggagaa
cctgagcccg ggtccccaaa 3300 gggacaagta gctgatagtg aggcagctga
gccccatggc ggcctgccca agtggcacgg 3360 gaaagtggag ctctctgctg
cccccactac tggccccatc tcttggctct cccctccctt 3420 cctcctgtgg
agaaggccca tctctggaaa ggcctcctag acatgcggca ctttgcaaag 3480
cctgtcgggc tcacagcccc tctagggtct aggaccttga gaatgaagaa tggagtcact
3540 tctagactct agtggtaacc accaggaggt acagggtgct ctgactgtgc
agggaaaccc 3600 accgtgggct ctgctgagcc aagtgcctgt gaggctggag
agtctggtgc ccttgttctg 3660 agatagcatc ttgctatgtt gccctcaagt
cccaggcaac tggggctgca ggagcaccac 3720 cttgcctctc tccagcttct
tgaagacttg tacctttctc ctagcagtct ctatctgctc 3780 tcactccatc
cattgagcag ctattagctt gtggccaagt attttccagg ccctgtactg 3840
agttttaggg tacaagtttg agaaaggaag ggtggggtcc ttgctcctgg tccgtgaatg
3900 atgttgatgg cagaaacgat agttacacta gatgctaagg gctgtgggta
tctagaggga 3960 gcagggagca tgtgggataa cctgagcagg cctagctgaa
aagtcattgc tggcatgaga 4020 ctgctccagt agtacaggct gggaacacac
atttgaatgt ttcctgaaga cagttgggag 4080 ccacaggaaa tatccactgt
agaaagatta tttagttgta agacagagta gtagattggt 4140 taacatagta
gcaaaaacgt ggccccagtt tttacagatg aagggaattg gaactcagag 4200
aggttaagta acttctccca agcagctcag ctacaaaaat cacagaacag gcaggggcct
4260 gatggctctg atgcctgtgc tggtcccact attccatgtt gctaattcct
gcagcagcag 4320 taaacctctg ccttgtggaa atgaggagtc taaataaaga
gaccatagca ttgccacaag 4380 caggtttcta ccaactgggg gtggcaagga
atgctgtgtt agcagcagga agctgggaag 4440 aggctgagta ctggggggat
gaggaaggga tccccaggag aggctgactt tggccttgaa 4500 gaatggtgga
gtccctggaa agatgcagat acacagagct ctgtggatat acagagaagt 4560
ggggagctaa gtaggtggct tggggccatc atgtgacaga ggaagtcggg ctagatgcag
4620 gaagcccggt gctgtggcct agggagccat gtaggttctt tgagcagggg
gcgggggggg 4680 gggggggtga cccaggagtg actgtaaaca acatcaggcc
atgagcagct ctgacctaat 4740 gttctcacca agggagccag aaccaaggct
tagagccctg tcccttttta gtgtccaagg 4800 tcactttact ggccctcttc
ctttacagct gttggccccc acaggccatc aggcacctat 4860 gctattttat
tttatagcct tcattacaat gactacaatt gtaattagag agttgacagg 4920
gtcacatctg tccttatata ttccccctct gctaagttct gcctgggaga atgtggaggg
4980 tattggtgaa atttggggaa gttataaccc ccccacccct gcccccaccc
cctgctttgc 5040 tccctttatc tgcagggcat ttctgtgccc actttagccc
atatagctcc caaataaatg 5100 acacagaaac ctggtatttt cattaacaaa
ctgctggcac tctgctgggc aggttctgag 5160 ctgttctaac cctctaagct
gctaatgccc agatagatgc cccaatgctt gccatccgag 5220 tctttctctg
gcttgctctg ctccatgtgt gacctcatgg tgaatcctcc tgatttcccc 5280
acatggcctc tccacacttt tccttctccc ctctctctac cagggaccct ctcactggga
5340 cccgatgtcc catctgtact gtcctctccc acccagtcat aggctgattg
agtctttatt 5400 aaccaatcag agatgatgga aaaacagttt ttacatagca
ctgaggatgg agatgcttga 5460 cccttgagat gcttgcccgt aacctgtact
gtatccagat gtctgggccc ccaaatcagc 5520 aggtgaatac acagtacaca
ggactgaccc ccaacagagg gggaacacag gttctcactc 5580 tgggctccac
gccctcggcc ctttcttagt gcaggggtta gactttgtat gtgttgatga 5640
tgaggtaagg gccatggaac agtcagaacg gtggtgtcag aatcctgtcc ctctccctcc
5700 tgtcctcatc cctccttacc gtgtcactct tctgtctgtt gcaggtggtt
tctggctggg 5760 tatagaccaa gaaggagctg agggaacctt gtccctgacg
ggcaccatct tcggggtgct 5820 ggccagcctc tgcgtctccc tcaatgccat
ctataccaag aaggtgctcc ctgcagtaga 5880 ccacagtatc tggcgcctaa
ccttctataa caatgtcaat gcctgcgtgc tcttcttgcc 5940 cctgatgata
gtgctgggcg agctccgtgc cctcctggcc ttcactcatc tgagcagtgc 6000
ccacttctgg ctcatgatga cgctgggtgg cctgtttggc tttgccatcg gctatgtgac
6060 aggactgcag atcaaattca ccagtcccct gacccataac gtgtcaggca
cggccaaggc 6120 ctgtgcacag acagtgctgg ccgtgctcta ctacgaagag
attaagagct tcctgtggtg 6180 gacaagcaac ctgatggtgc tgggtggctc
ctccgcctac acctgggtca ggggctggga 6240 gatgcagaag acccaggagg
accccagctc caaagatggt gagaagagtg ctatcagggt 6300 gtgagctcct
tcagggagcc agggctgagc tcgggtgggg cctgcccagc acggaaggct 6360
tcccatagag cctactgggt atggccctga gcaataatgt ttacatcctt ctcagaagac
6420 catctaagaa gagccaggtt ctttcctgat aatgtcagaa agctgccaaa
tctcctgcct 6480 gccccatctt ctagtcttgg gaaagcccta ccaggagtgg
cacccttcct gcctcctcct 6540 ggggcctgtc tacctccata tggtctctgg
ggttggggcc agctgcactc tttgggcact 6600 ggactgatga agtgatgtct
tactttctac acaagggaga tgggttgtga ccctactata 6660 gctagttgaa
gggagctgtg taaccccaca tctctggggc cctgggcagg tagcataata 6720
gctaggtgct attaacatca ataacacttc agactacctt tggaggcagt tgggagctga
6780 gccgagagag agagatggcc attctgccct cttctgtgtg gatgggtatg
acagaccaac 6840 tgtccatggg gtgactgaca cctccacact tcatattttc
aactttagaa aagggggagc 6900 cacacgtttt acagattaag tggagtgatg
aatgcctcta cagcccctaa ccccactttc 6960 cctgcctggc ttctcttggc
ccagaagggc caccatcctg ttctccaaca cctgacccag 7020 ctatctggct
atactctctt tctgtactcc cttccccttc ccccccccat tagcctcctc 7080
cccaacacct ccatcttcag gcaggaagtg gggtccactc agcctctgtt cccatctgct
7140 tggacccctg agcctctcat gaaggtaggc ttatgttctc tgaggctggg
gccggaggag 7200 cgcactgatt ctcggagtta tcccatcagg ctcctgtcac
aaaatagcct aggccgtgtg 7260 tctaagaaca gtggaggttg gcttataact
gttctggggg cagcgaagcc cacatcaagg 7320 tactcataga cccagtattt
ctgaggaaac ccctgtccac atcctcactt ggtaaaggca 7380 cagataatct
ccctcaggcc tcttgtatag gagcactagc cctgggaggg ctccgcccca 7440
tgacctgatc accccaaagc cttcaacaga aggattccaa catgaatttg gggacagaag
7500 cactcagacc acgatgccca gcaccacacc ctcctatcct cagggtagct
gtcactgtcc 7560 tagtcccttc tgtttggcct tttgtaccct catttccttg
gcgtcatgtt tgatgtcttt 7620 gtctctttcg tgagaagatg gggaaaccat
gtcagcctct gcttccgact tcccatgggt 7680 tctaatgaag ttggtggggc
ctgatgccct gagttgtatg tgatttaata aaaaaaaaat 7740 ttttttaaaa ac 7752
100 4039 DNA Cricetulus griseus 100 gaacttcacc caagccatgt
gacaattgaa ggctgtaccc ccagacccta acatcttgga 60 gccctgtaga
ccagggagtg cttctggccg tggggtgacc tagctcttct accaccatga 120
acagggcccc tctgaagcgg tccaggatcc tgcgcatggc gctgactgga ggctccactg
180 cctctgagga ggcagatgaa gacagcagga acaagccgtt tctgctgcgg
gcgctgcaga 240 tcgcgctggt cgtctctctc tactgggtca cctccatctc
catggtattc ctcaacaagt 300 acctgctgga cagcccctcc ctgcagctgg
atacccctat cttcgtcact ttctaccaat 360 gcctggtgac ctctctgctg
tgcaagggcc tcagcactct ggccacctgc tgccctggca 420 ccgttgactt
ccccaccctg aacctggacc ttaaggtggc ccgcagcgtg ctgccactgt 480
cggtagtctt cattggcatg ataagtttca ataacctctg cctcaagtac gtaggggtgg
540 ccttctacaa cgtggggcgc tcgctcacca ccgtgttcaa tgtgcttctg
tcctacctgc 600 tgctcaaaca gaccacttcc ttctatgccc tgctcacatg
tggcatcatc attggtgagt 660 ggggcccggg ggctgtggga gcaggatggg
catcgaactg aagccctaaa ggtcaacact 720 gtaggtacct ttacttactg
tcccaggtcc cttgcatcag cagttacagg aagagccctg 780 tagaaaacaa
ataacttcct tatggtcatt caacaagtta gggacccagc cagggtgaaa 840
ataatgttag cagcaactac agcaaagatg gctctcgcca cttgcatgat taaaatgtgc
900 caggtactca gatcyaagca ttggatccac attaactcaa ctaatcccta
ttacaaggta 960 aaatatatcc gaattttaca gagggaaaac caaggcacag
agaggctaag tagcttgacc 1020 aggatcacac agctaataat cactgacata
gctgggattt aaacataagc agttacctcc 1080 atagatcaca ctatgaccac
catgccactg ttccttctca agagttccag gatcctgtct 1140 gtccagttct
ctttaaagag gacaacacat ctgacattgc taccttgagg taacatttga 1200
aatagtgggt agacatatgt tttaagtttt attcttrctt tttatgygtg tgtgtttggg
1260 gggccaccac agtgtatggg tggagataag gggacaactt aagaattggt
cctttctccc 1320 accacatggg tgctgaggtc tgaactcagg tcatcaggat
tggcacaaat ccctttaccc 1380 actgagccat
ttcactggtc caatatatgt gtgcttttaa gaggctttaa ctattttccc 1440
agatgtgaat gtcctgctga tcattatccc cttttacccg gaagccctct gggaggtgcc
1500 atccctgtgg tcgtctgcat acaaatgggg aaactgcaac tcagagaaac
aaggctactt 1560 gccagggccc cacaagtaag ataggctggg atgccatccc
agactggcca cactccctgg 1620 cctgtgcttc aagccagttt actttgttcc
tgcccattgg aagttagcat gttgcagtca 1680 aacacaataa ctacaggcca
aaagtgcttt taaattaaag tcagatgaac ttttaaacat 1740 ccagagctcc
tcaactgcag gagttacaac ctgattctgc aaccatcttt gcagtgcccg 1800
gtagtcatat gtagctagag gctcttggct aggacagcat gtgttaggaa acatctggcc
1860 ctgagatcat tgaattgagt gactgctggg tgacaaagac caaggcatcc
gttccctgag 1920 agtcctgggc aagcagcaat gtgaccttca tttgtaccta
ctcaggttct ttatctgtcc 1980 tgtttgacct acttagtctc ctctggtgtc
tcagaggccc aggctgggta ctctggatgt 2040 caggatcagg ccaatgcgca
catctgccct agaaatgtcc ccctggttga gcagctcctg 2100 aatccatcgg
taaagggtct ggaccaggga ggagtcagat aaaaagctga cagcactggg 2160
ggactccatg gggaactccc acctgccccc acacatccat cctaagagaa ctggtattcc
2220 ttgtttcctc tttgtcctac aaggcaccct gggatcccac ttcagtctcc
cagccttgcc 2280 agggttagag ggcatgagcc tccttgtggg gaatttagat
gcaagaaggt acagtcacta 2340 gagaacctga gctcagatcc ccaaagtaac
cagtacctga tagtgaggca gctgagaacc 2400 gcagcagcct gcctgagtgg
ctgaactctg cggcctccgg aactggcccc aactgttggg 2460 tctcctcttc
cttcctcctg tgagggaggg cccatctctg ataagtgctg tggggactct 2520
agagtaggga ggaggaggag caatctaagc aggccttact gagaagtcct tgctggcatg
2580 tggctgcctg aggagtacag actgggaaca cccatttgaa tgagtaaggt
ttttcctgaa 2640 ggccatgggg agccacggag gaaaatcatt ttagttacaa
gacaaagagt agattggtta 2700 acatgggagc arggacatgg ccccaatttt
cattgatgaa ggaaattgga actcrgagag 2760 gttaagtaac ttctcccaaa
tagctcagct tcataatcac agaacagtca gagtctagat 2820 ctctctgatg
cctgtgatgg tcctgccatt ccatgttgct gatccctgtg gcatcagtaa 2880
gcctctacct tgtgggaatg caggatctaa atgaagagag raagtgctgg ccccatgctg
2940 tggtctggaa agctatgcag gctctttgag cagagagtga cccacaagtg
aatagagtcc 3000 tatgagactc aaagcaacat ccacccttaa gcagctctaa
ccaaatgctc acactgaggg 3060 agccaaagcc aagttagagt cctgtgcttg
cccaaggtca ctttgcctgg ccctcctcct 3120 atagcacccg tgttatctta
tagccctcat tacagtgatt acaattataa ttagagaggt 3180 aacagggcca
cactgtcctt acacattccc ctgctagatt gtagctggga gagggggaga 3240
tgtaggtggc tgggggagtg ggagggaaga tgcagatttt cattctgggc tctactccct
3300 cagccatttt ttggtgtggg agttagactt tggatatgtt gatgatgagg
taagggccac 3360 agaacagtct gaactgtggt atcagaatcc tgtccctctc
cctctctcct catccctctt 3420 caccttgtca ctcctctgtc tgctacaggt
ggtttctggc tgggtataga ccaagaggga 3480 gctgagggca ccctgtccct
cataggcacc atcttcgggg tgctggccag cctctgcgtc 3540 tccctcaatg
ccatctatac caagaaggtg ctcccagcag tggacaacag catctggcgc 3600
ctaaccttct ataacaatgt caatgcctgt gtgctcttct tgcccctgat ggttctgctg
3660 ggtgagctcc gtgccctcct tgactttgct catctgtaca gtgcccactt
ctggctcatg 3720 atgacgctgg gtggcctctt cggctttgcc attggctatg
tgacaggact gcagatcaaa 3780 ttcaccagtc ccctgaccca caatgtatca
ggcacagcca aggcctgtgc gcagacagtg 3840 ctggccgtgc tctactatga
agagactaag agcttcctgt ggtggacaag caacctgatg 3900 gtgctgggtg
gctcctcagc ctatacctgg gtcaggggct gggagatgca gaagacccaa 3960
gaggacccca gctccaaaga gggtgagaag agtgctattg gggtgtgagc ttcttcaggg
4020 acctgggact gaacccaag 4039
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